EP3914355A1 - Arn thérapeutique et anticorps anti-pd1 pour des cancers à tumeurs solides de stade avancé - Google Patents

Arn thérapeutique et anticorps anti-pd1 pour des cancers à tumeurs solides de stade avancé

Info

Publication number
EP3914355A1
EP3914355A1 EP20705267.1A EP20705267A EP3914355A1 EP 3914355 A1 EP3914355 A1 EP 3914355A1 EP 20705267 A EP20705267 A EP 20705267A EP 3914355 A1 EP3914355 A1 EP 3914355A1
Authority
EP
European Patent Office
Prior art keywords
tumor
cancer
seq
subject
rna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP20705267.1A
Other languages
German (de)
English (en)
Inventor
Serena MASCIARI
Semra YORUK
Karl Hsu
Nicolas ACQUAVELLA
Marie BERNARDO
Robert JABULOWSKY
Ugur Sahin
Friederike GIESEKE
Zuzana JIRAKOVA TRNKOVA
Timothy R. WAGENAAR
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sanofi SA
Original Assignee
Sanofi SA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sanofi SA filed Critical Sanofi SA
Publication of EP3914355A1 publication Critical patent/EP3914355A1/fr
Pending legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7115—Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/193—Colony stimulating factors [CSF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/208—IL-12
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2086—IL-13 to IL-16
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A61K38/212—IFN-alpha
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00

Definitions

  • This disclosure relates to the field of therapeutic RNA to treat solid tumor cancers, including, for example, in subjects that have failed, or become intolerant, resistant, or refractory to an anti-programmed cell death 1 (PD-1) or anti-programmed cell death 1 ligand (PD-L1) therapy, including subjects with acquired or innate resistance to an anti programmed cell death 1 (PD-1) or anti-programmed cell death 1 ligand (PD-L1) therapy, and subjects with advanced- stage or metastatic solid tumors.
  • PD-1 anti-programmed cell death 1
  • PD-L1 anti-programmed cell death 1 ligand
  • Solid tumors as abnormal masses of tissue that do not normally contain cysts or liquid areas. Solid tumors can be physically located in any tissue or organ including the ovary, breast, colon, and other tissues, and include melanoma, cutaneous squamous cell cancer (CSCC), squamous cell carcinoma of the head and neck (HNSCC), non-small cell lung cancer, kidney cancer, head and neck cancer, thyroid cancer, colon cancer, liver cancer, ovarian cancer, breast cancer.
  • CSCC cutaneous squamous cell cancer
  • HNSCC squamous cell carcinoma of the head and neck
  • non-small cell lung cancer kidney cancer, head and neck cancer
  • thyroid cancer colon cancer
  • liver cancer ovarian cancer
  • breast cancer ovarian cancer
  • Immune checkpoint blockade such as with anti -PD-1 and anti-PD-Ll therapy elicits anticancer responses in the clinic, however a large proportion of patients do not benefit from treatment.
  • Several mechanisms of innate and acquired resistance to checkpoint blockade have been defined and include mutations of MHC I and IFNy signaling pathways.
  • Advanced stage solid tumor cancers are particularly difficult to treat.
  • Current treatments include surgery, radiotherapy, immunotherapy and chemotherapy.
  • Surgery alone may be an appropriate treatment for small localized tumors, but large invasive tumors may be unresectable by surgery.
  • Other common treatments such as radiotherapy and chemotherapy are associated with undesirable side effects and damage to healthy cells.
  • compositions, uses, and methods that can overcome present shortcomings in treatment of solid tumors, such as advanced-stage, unresectable, or metastatic solid tumor cancers, including in subjects that have failed, or become intolerant, resistant, or refractory to an anti-programmed cell death 1 (PD-1) or anti-programmed cell death 1 ligand (PD-L1) therapy.
  • PD-1 anti-programmed cell death 1
  • PD-L1 anti-programmed cell death 1 ligand
  • Administration of therapeutic RNAs as disclosed herein can reduce tumor size, prolong time to progressive disease, and/or protect against metastasis and/or recurrence of the tumor and ultimately extend survival time.
  • RNAs comprising RNA encoding an IL-12sc protein, RNA encoding an IL-15 sushi protein, RNA encoding an IFNa protein, and RNA encoding a GM-CSF protein, and administering an effective amount of an anti-programmed cell death 1 (PD-1) antibody, wherein the subject has failed, or become intolerant, resistant, or refractory to an anti-programmed cell death 1 (PD-1) or anti programmed cell death 1 ligand (PD-L1) therapy.
  • PD-1 anti-programmed cell death 1
  • methods of treating a solid tumor cancer in a subject that has failed, or become intolerant, resistant, or refractory to an anti-programmed cell death 1 (PD-1) or anti-programmed cell death 1 ligand (PD-L1) therapy comprising administering an effective amount of RNAs comprising RNA encoding an IL-12sc protein, RNA encoding an IL-15 sushi protein, RNA encoding an IFNa protein, and RNA encoding a GM-CSF protein and administering an effective amount of an anti-programmed cell death 1 (PD-1) antibody, to a subject that has failed, or become intolerant, resistant, or refractory to an anti-programmed cell death 1 (PD-1) or anti-programmed cell death 1 ligand (PD-L1) therapy.
  • RNAs comprising RNA encoding an IL-12sc protein, RNA encoding an IL-15 sushi protein, RNA encoding an IFNa protein, and RNA encoding a GM-CSF
  • Methods of treating a subject having anti -PD-1 and/or anti-PD-Ll resistant solid tumor cancer comprising administering an effective amount of RNAs comprising RNA encoding an IL-12sc protein, RNA encoding an IL-15 sushi protein, RNA encoding an IFNa protein, and RNA encoding a GM-CSF protein and administering an effective amount of an anti-programmed cell death 1 (PD-1) antibody to a subject that has an anti-PD-1 and/or anti-PD-Ll resistant solid tumor cancer.
  • RNAs comprising RNA encoding an IL-12sc protein, RNA encoding an IL-15 sushi protein, RNA encoding an IFNa protein, and RNA encoding a GM-CSF protein and administering an effective amount of an anti-programmed cell death 1 (PD-1) antibody to a subject that has an anti-PD-1 and/or anti-PD-Ll resistant solid tumor cancer.
  • PD-1 anti-programmed cell death 1
  • RNAs comprising RNA encoding an IL-12sc protein, RNA encoding an IL-15 sushi protein, RNA encoding an IFNa protein, and RNA encoding a GM-CSF protein and administering an effective amount of an anti-programmed cell death 1 (PD-1) antibody to a subject that has a solid tumor cancer with acquired resistance to anti- PD-1 and/or anti-PD-Ll therapy.
  • RNAs comprising RNA encoding an IL-12sc protein, RNA encoding an IL-15 sushi protein, RNA encoding an IFNa protein, and RNA encoding a GM-CSF protein and administering an effective amount of an anti-programmed cell death 1 (PD-1) antibody to a subject that has a solid tumor cancer with acquired resistance to anti- PD-1 and/or anti-PD-Ll therapy.
  • PD-1 anti-programmed cell death 1
  • methods of treating a subject having a solid tumor cancer with innate resistance to anti-PD-1 and/or anti-PD-Ll therapy comprising administering an effective amount of RNAs comprising RNA encoding an IL- 12sc protein, RNA encoding an IL-15 sushi protein, RNA encoding an IFNa protein, and RNA encoding a GM-CSF protein and administering an effective amount of an anti- programmed cell death 1 (PD-1) antibody to a subject that has a solid tumor cancer with innate resistance to anti-PD-1 and/or anti-PD-Ll therapy.
  • RNAs comprising RNA encoding an IL- 12sc protein, RNA encoding an IL-15 sushi protein, RNA encoding an IFNa protein, and RNA encoding a GM-CSF protein and administering an effective amount of an anti- programmed cell death 1 (PD-1) antibody to a subject that has a solid tumor cancer with innate resistance to anti-PD-1 and/or anti-PD-Ll therapy
  • Embodiments provided herein are not limited by any scientific theory regarding intolerance, resistance, or refraction.
  • the intolerance, resistance, refraction (including acquired and innate resistance) to an anti-PD-1 and/or anti-PD-1 therapy results from a cancer cell comprising a partial or total loss of beta-2-microglobulin (B2M) function.
  • a subject has a cancer cell comprising a partial or total loss of beta-2- microglobulin (B2M) function.
  • the cancer cell has a partial loss of B2M function.
  • the cancer cell has a total loss of B2M function.
  • the partial or total loss of B2M function is assessed by comparing a cancer cell to a non-cancer cell from the same subject, optionally wherein the non-cancer cell is from the same tissue from which the cancer cell was derived.
  • the cancer cell is in a solid tumor that comprises cancer cells with normal B2M function.
  • the cancer cell is in a solid tumor in which 25% or more of the cancer cells have a partial or total loss in B2M function.
  • the cancer cell is in a solid tumor in which 50% or more of the cancer cells have a partial or total loss in B2M function.
  • the cancer cell is in a solid tumor in which 75% or more of the cancer cells have a partial or total loss in B2M function. In some embodiments, the cancer cell is in a solid tumor in which 95% or more of the cancer cells have a partial or total loss in B2M function. In some embodiments, the solid tumor as a whole ( e.g ., as assessed in a biopsy taken from the solid tumor) has a partial or total loss of B2M function compared to normal cells or tissue from which the solid tumor is derived. In some embodiments, the subject comprises (e.g., the partial or total loss of function results from) a mutation in the B2M gene. The mutation may be a substitution, insertion, or deletion. In some embodiments, the B2M gene comprises a loss of heterozygosity (LOH).
  • LHO loss of heterozygosity
  • the mutation is a frameshift mutation.
  • the frameshift mutation is in exon 1 of B2M.
  • the frameshift mutation comprises p.Leul3fs and/or p.Serl4fs.
  • the subject has a reduced level of B2M protein as compared to a subject without a partial or total loss of B2M function.
  • the solid tumor e.g, cancer cells within the solid tumor
  • a solid tumor sample e.g, a biopsy comprising cancer cells of the solid tumor
  • the level of MHC I expressed on the surface of cancer cells in the solid tumor is reduced as a result of a mutation in a B2M gene.
  • a subject has a cancer cell comprising a reduced level of surface expressed MHC I.
  • the cancer cell has no surface expressed MHC I.
  • the reduced level of surface expressed MHC I is assessed by comparing a cancer cell to a non-cancer cell from the same subject, optionally wherein the non-cancer cell is from the same tissue from which the cancer cell was derived.
  • the cancer cell is in a solid tumor that comprises cancer cells with a normal level of surface expressed MHC I.
  • the cancer cell is in a solid tumor in which 25% or more of the cancer cells have a reduced level of surface expressed MHC I.
  • the cancer cell is in a solid tumor in which 50% or more of the cancer cells have a reduced level of surface expressed MHC I. In some embodiments, the cancer cell is in a solid tumor in which 75% or more of the cancer cells have a reduced level of surface expressed MHC I. In some embodiments, the cancer cell is in a solid tumor in which 95% or more of the cancer cells have a reduced level of surface expressed MHC I. In some embodiments, the solid tumor as a whole (e.g, as assessed in a biopsy taken from the solid tumor) has a reduced level of surface expressed MHC I compared to normal cells or tissue from which the solid tumor is derived.
  • methods for treating a subject having an advanced- stage, unresectable, or metastatic solid tumor cancer comprising administering an effective amount of RNAs comprising RNA encoding an IL-12sc protein, RNA encoding an IL-15 sushi protein, RNA encoding an IFNa protein, and RNA encoding a GM-CSF protein and administering an effective amount of an anti-programmed cell death 1 (PD-1) antibody to a subject that has an advanced-stage, unresectable, or metastatic solid tumor cancer.
  • RNAs comprising RNA encoding an IL-12sc protein, RNA encoding an IL-15 sushi protein, RNA encoding an IFNa protein, and RNA encoding a GM-CSF protein and administering an effective amount of an anti-programmed cell death 1 (PD-1) antibody to a subject that has an advanced-stage, unresectable, or metastatic solid tumor cancer.
  • PD-1 anti-programmed cell death 1
  • the subject has failed, or become intolerant, resistant, or refractory to an anti-programmed cell death 1 (PD-1) therapy. In some embodiments, the subject has failed, or become intolerant, resistant, or refractory to an anti-programmed cell death 1 ligand (PD-L1) therapy.
  • PD-1 anti-programmed cell death 1
  • PD-L1 anti-programmed cell death 1 ligand
  • the subject has failed an anti -programmed cell death 1
  • PD-1 therapy or anti-programmed cell death 1 ligand (PD-L1) therapy.
  • the subject has become intolerant to an anti programmed cell death 1 (PD-1) or anti-programmed cell death 1 ligand (PD-L1) therapy.
  • PD-1 programmed cell death 1
  • PD-L1 anti-programmed cell death 1 ligand
  • the subject has become resistant to an anti -programmed cell death 1 (PD-1) and/or anti-programmed cell death 1 ligand (PD-L1) therapy.
  • PD-1 anti -programmed cell death 1
  • PD-L1 anti-programmed cell death 1 ligand
  • the subject has become refractory to an anti programmed cell death 1 (PD-1) or anti-programmed cell death 1 ligand (PD-L1) therapy.
  • PD-1 programmed cell death 1
  • PD-L1 anti-programmed cell death 1 ligand
  • the refractory or resistant cancer is one that does not respond to a specified treatment.
  • the refraction occurs from the very beginning of treatment. In some embodiments, the refraction occurs during treatment.
  • the cancer is resistant before treatment begins.
  • the subject has a cancer that does not respond to the anti-programmed cell death 1 (PD-1) and/or anti-programmed cell death 1 ligand (PD-L1) therapy.
  • the subject has a cancer that is becoming refractory or resistant to a specified treatment.
  • the specified treatment is as an anti -PD 1 therapy.
  • the specified treatment is as an anti-PD-Ll therapy.
  • the subject has become less responsive to the therapy since first receiving it.
  • the subject has not received the therapy, but has a type of cancer that does not typically respond to the therapy.
  • the subject is human.
  • the subject has not been treated previously with an anti-PD-1 or anti-PD-Ll therapy.
  • the solid tumor cancer is one in which an anti-PD-1 or anti-PD-Ll therapy is not routinely used.
  • the subject has a metastatic solid tumor. In some embodiments, the subject has a non-metastatic solid tumor. In some embodiments, the subject has an unresectable solid tumor. In some embodiments, the subject has a metastatic and unresectable solid tumor. In some embodiments, the subject has a non-metastatic and unresectable solid tumor.
  • the solid tumor is an epithelial tumor, prostate tumor, ovarian tumor, renal cell tumor, gastrointestinal tract tumor, hepatic tumor, colorectal tumor, tumor with vasculature, mesothelioma tumor, pancreatic tumor, breast tumor, sarcoma tumor, lung tumor, colon tumor, melanoma tumor, small cell lung tumor, neuroblastoma tumor, testicular tumor, carcinoma tumor, adenocarcinoma tumor, seminoma tumor, retinoblastoma, cutaneous squamous cell carcinoma (CSCC), squamous cell carcinoma for the head and neck (HNSCC), head and neck cancer, osteosarcoma tumor, cutaneous squamous cell cancer (CSCC), non-small cell lung cancer, kidney tumor, thyroid tumor, liver tumor, or other solid tumors amenable to intratumoral injection.
  • CSCC cutaneous squamous cell carcinoma
  • HNSCC head and neck cancer
  • osteosarcoma tumor cutaneous squamous cell
  • the solid tumor is a lymphoma, including Non-Hodgkin lymphoma or Hodgkin lymphoma.
  • the solid tumor cancer is melanoma.
  • the melanoma is uveal melanoma or mucosal melanoma. In some embodiments,
  • the solid tumor cancer is melanoma, optionally uveal melanoma or mucosal melanoma, and comprises superficial, subcutaneous and/or lymph node metastases amenable for intratumoral injection.
  • intratumoral injection comprises injection into a solid tumor metastasis within a lymph node. In some embodiments, intratumoral injection comprises injection into a lymphoma tumor within a lymph node. In some embodiments, intratumoral injection comprises injection into a primary or secondary solid tumor that is within 10 cm of the subject’s skin surface. In some embodiments, intratumoral injection comprises injection into a primary or secondary solid tumor that is within 5 cm of the subject’s skin surface. In some embodiments, intratumoral injection comprises injection into a cutaneous solid tumor. In some embodiments, the cutaneous solid tumor is a metastasis. In some embodiments, the cutaneous solid tumor is a skin cancer. In some embodiments, the cutaneous solid tumor is not a skin cancer.
  • intratumoral injection comprises injection into a subcutaneous solid tumor.
  • the subcutaneous solid tumor is a metastasis.
  • the subcutaneous solid tumor is a skin cancer. In some embodiments, the subcutaneous solid tumor is not a skin cancer.
  • the solid tumor is an epithelial tumor. In some embodiments, the solid tumor is a prostate tumor. In some embodiments, the solid tumor is an ovarian tumor. In some embodiments, the solid tumor is a renal cell tumor. In some embodiments, the solid tumor is a gastrointestinal tract tumor. In some embodiments, the solid tumor is a hepatic tumor. In some embodiments, the solid tumor is a colorectal tumor.
  • the solid tumor is a tumor with vasculature. In some embodiments, the solid tumor is a mesothelioma tumor. In some embodiments, the solid tumor is a pancreatic tumor. In some embodiments, the solid tumor is a breast tumor. In some embodiments, the solid tumor is a sarcoma tumor. In some embodiments, the solid tumor is a lung tumor. In some embodiments, the solid tumor is a colon tumor. In some embodiments, the solid tumor is a melanoma tumor. In some embodiments, the solid tumor is a small cell lung tumor. In some embodiments, the solid tumor is non-small cell lung cancer tumor. In some
  • the solid tumor is a neuroblastoma tumor. In some embodiments, the solid tumor is a testicular tumor. In some embodiments, the solid tumor is a carcinoma tumor. In some embodiments, the solid tumor is an adenocarcinoma tumor. In some embodiments, the solid tumor is a seminoma tumor. In some embodiments, the solid tumor is a retinoblastoma. In some embodiments, the solid tumor is a cutaneous squamous cell carcinoma (CSCC). In some embodiments, the solid tumor is a squamous cell carcinoma for the head and neck (HNSCC). In some embodiments, the solid tumor is HNSCC. In some embodiments, the solid tumor is head and neck cancer. In some embodiments, the solid tumor is an cutaneous squamous cell carcinoma (CSCC). In some embodiments, the solid tumor is a squamous cell carcinoma for the head and neck (HNSCC). In some embodiments, the solid tumor is HNSCC. In some embodiments, the solid tumor is head and neck
  • the solid tumor is kidney cancer. In some embodiments, the solid tumor is thyroid cancer. In some embodiments, the solid tumor is anaplastic thyroid cancer (ATC). In some embodiments, the solid tumor is liver cancer. In some embodiments, the solid tumor is a colon tumor. In some embodiments, the solid tumor is any two of the above. In some embodiments, the solid tumor is any two or more of the above.
  • the solid tumor is lymphoma. In some embodiments, the solid tumor is Non-Hodgkin lymphoma. In some embodiments, the solid tumor is Hodgkin lymphoma. In some embodiments, the solid tumor lymphoma is not a central nervous system lymphoma. [0033] In some embodiments, the solid tumor cancer is HNSCC. In some embodiments,
  • the solid tumor cancer is mucosal melanoma with only mucosal sites. In some embodiments, the solid tumor cancer is HNSCC and mucosal melanoma with only mucosal sites.
  • the solid tumor cancer is uveal melanoma or mucosal melanoma. In some embodiments, the solid tumor cancer is breast cancer. In some embodiments, the solid tumor cancer is breast sarcoma or triple negative breast cancer.
  • the RNAs are administered in combination with an anti-PD-1 antibody.
  • the subject has more than one solid tumor.
  • at least one tumor is resistant, refractory, or intolerant to PD-1 or PD-L1 therapy.
  • at least one tumor is resistant, refractory, or intolerant to PD-1 or PD- L1 therapy and at least one tumor is not.
  • both resistant and non-resistant tumors, if present, are successfully treated.
  • the solid tumor cancer is stage III, subsets of stage III, stage IV, or subsets of stage IV. In some embodiments, the solid tumor cancer is stage IIIB, stage IIIC, or stage IV cancer.
  • the solid tumor cancer is advanced-stage. In some embodiments, the solid tumor cancer is unresectable. In some embodiments, the solid tumor cancer is advanced-stage and unresectable.
  • the solid tumor has spread from its origin to another site in the subject.
  • the solid tumor cancer has one or more cutaneous or subcutaneous lesions. In some embodiments, the solid tumor cancer has metastasized. In some embodiments, the solid tumor cancer has metastasized, but is not a skin cancer.
  • the subject is without other treatment options.
  • the solid tumor cancer is one for which an anti -PD 1 or anti-PD-Ll therapy is routinely used, but which has not been treated with the therapy yet.
  • the solid tumor cancer is stage IIIB, IIIC, or unresectable stage IV melanoma that is resistant and/or refractory to anti-PD-1 or anti-PD-Ll therapy.
  • the solid tumor cancer comprises superficial or subcutaneous lesions and/or metastases.
  • the subject has measurable disease according to the
  • the subject has a life expectancy of more than 3 months. In some embodiments, the subject is at least 18 years of age.
  • the RNAs are injected intratum orally.
  • the RNAs are injected intratum orally only at mucosal sites of the solid tumor cancer.
  • the RNAs are administered for about 5 months. In some embodiments, the RNAs are administered once every week. In some embodiments, the RNAs are administered for a maximum of 52 weeks.
  • the IFNa protein is an IFNa2b protein.
  • the RNA encoding an IL-12sc protein comprises the nucleotide sequence of SEQ ID NO: 17 or 18, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 17 or 18; and/or the IL-12sc protein comprises the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 14; and/or the RNA encoding an IL-12sc protein comprises a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the p40 portion of IL-12sc (nucleotides 1-984 of SEQ ID NO: 17 or 18) and at least 99%, 98%, 97%, 96%, 95%, 90%
  • the RNA encoding an IL-15 sushi protein comprises the nucleotide sequence of SEQ ID NO: 26, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 26; and/or the IL-15 sushi protein comprises the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 24; and/or the RNA encoding an IL-15 sushi protein comprises a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the sushi domain of IL-15 receptor alpha (nucleotides 1-321 of SEQ ID NO: 26) and at least 99%, 98%, 97%, 96%, 95%, 90%,
  • the RNA encoding an IFNa protein comprises the nucleotide sequence of SEQ ID NO: 22 or 23, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 22 or 23 and/or the IFNa protein comprises the amino acid sequence of SEQ ID NO: 19, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 19.
  • the RNA encoding a GM-CSF protein comprises the nucleotide sequence of SEQ ID NO: 29, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 29 and/or the GM-CSF protein comprises the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 27.
  • At least one RNA comprises a modified nucleoside in place of at least one uridine. In some embodiments, at least one RNA comprises a modified nucleoside in place of each uridine. In some embodiments, each RNA comprises a modified nucleoside in place of at least one uridine. In some embodiments, each RNA comprises a modified nucleoside in place of each uridine. In some embodiments, the modified nucleoside is independently selected from pseudouridine (y), N1 -methyl-pseudouridine (m 1 y), and 5- methyl-uridine (m5U).
  • At least one RNA comprises more than one type of modified nucleoside, wherein the modified nucleosides are independently selected from pseudouridine (y), N1 -methyl-pseudouridine (m 1 y), and 5-methyl-uridine (m5U).
  • the modified nucleoside is N1 -methyl-pseudouridine (ih ⁇ y).
  • At least one RNA comprises the 5’ cap m2 7 ’ 3
  • each RNA comprises the 5’ cap m2 7 ’ 3 °Gppp(mi 2 °)ApG (also sometimes referred to as m2 7 ’ 3 0 G(5’)ppp(5’)m 2 °ApG).
  • At least one RNA comprises a 5’ UTR comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 4 and 6, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 4 and 6.
  • each RNA comprises a 5’ UTR comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 4 and 6, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 4 and 6.
  • At least one RNA comprises a 3’ UTR comprising the nucleotide sequence of SEQ ID NO: 8, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 8.
  • each RNA comprises a 3’ UTR comprising the nucleotide sequence of SEQ ID NO: 8, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 8.
  • At least one RNA comprises a poly-A tail.
  • each RNA comprises a poly-A tail.
  • the poly-A tail comprises at least 100 nucleotides.
  • the poly-A tail comprises or consists of the poly-A tail shown in SEQ ID NO: 30.
  • one or more RNA comprises:
  • a 5’ cap comprising m27,3’-0Gppp(ml2’-0)ApG or 3'-0-Me- m7G(5')ppp(5')G;
  • a 5’ UTR comprising (i) a nucleotide sequence selected from the group consisting of SEQ ID NOs: 4 and 6, or (ii) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 4 and 6;
  • a 3’ UTR comprising (i) the nucleotide sequence of SEQ ID NO: 8, or (ii) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:8; and iv. a poly-A tail comprising at least 100 nucleotides.
  • the poly-A tail comprises or consists of SEQ ID NO:
  • treating the solid tumor comprises reducing the size of a tumor or preventing cancer metastasis in a subject.
  • the RNAs are administered at the same time. In some embodiments, the RNAs are administered via injection. In some embodiments, the RNAs are mixed together in liquid solution prior to injection.
  • the anti -PD 1 antibody is cemiplimab, pembrolizumab, nivolumab, MEDI0608, PDR001, PF-06801591, BGB-A317, pidilizumab, TSR-042, AGEN- 2034, A-0001, BGB-108, BI-754091, CBT-501, ENUM-003, ENUM-388D4, IBI-308, JNJ- 63723283, JS-001, JTX-4014, JY-034, CLA-134, STIA-1110, 244C8, or 388D4.
  • the anti-PDl antibody is cemiplimab.
  • the anti-PDl antibody is administered at a dose of about 0.1-600 mg. In some embodiments, the anti-PDl antibody is administered at a dose of 200 mg. In some embodiments, the anti-PDl antibody is administered at a dose of 240 mg. In some embodiments, the anti-PDl antibody is administered at a dose of 350 mg. In some embodiments, the anti-PDl antibody is administered via injection. In some embodiments, the anti-PDl antibody is administered intravenously. In some embodiments, the anti -PD- 1 antibody is administered once every three weeks. In some embodiments, the RNAs and the anti-PD-1 antibody are administered for about 8 months.
  • Embodiment A A composition comprising RNA encoding an IL-12sc protein, RNA encoding an IL-15 sushi protein, RNA encoding an IFNa protein, and RNA encoding a GM-CSF protein for use in treating a subject having a solid tumor cancer, in combination with an anti-programmed cell death 1 (PD-1) antibody, wherein the subject has failed, or become intolerant, resistant, or refractory to an anti-programmed cell death 1 (PD-1) or anti-programmed cell death 1 ligand (PD-L1) therapy.
  • PD-1 anti-programmed cell death 1
  • Embodiment A 2 A composition comprising RNA encoding an IL-12sc protein for use in treating a subject that has failed, or become intolerant, resistant, or refractory to an anti-programmed cell death 1 (PD-1) or anti programmed cell death 1 ligand (PD-L1) therapy, in combination with an anti-programmed cell death 1 (PD-1) antibody, wherein the RNA is co-administered with RNA encoding an IL-15 sushi, RNA encoding an IFNa protein, and RNA encoding a GM-CSF protein.
  • PD-1 anti-programmed cell death 1
  • P-L1 anti programmed cell death 1 ligand
  • Embodiment A 3 A composition comprising RNA encoding an IL-15 sushi protein for use in treating a subject that has failed, or become intolerant, resistant, or refractory to an anti-programmed cell death 1 (PD-1) or anti programmed cell death 1 ligand (PD-L1) therapy, in combination with an anti-programmed cell death 1 (PD-1) antibody, wherein the RNA is co-administered with RNA encoding an IL-12sc protein, RNA encoding an IFNa protein, and RNA encoding a GM-CSF protein.
  • PD-1 anti-programmed cell death 1
  • P-L1 anti programmed cell death 1 ligand
  • Embodiment A 4 A composition comprising RNA encoding an IFNa protein for use in treating a subject that has failed, or become intolerant, resistant, or refractory to an anti-programmed cell death 1 (PD-1) or anti
  • PD-1 anti-programmed cell death 1
  • RNA programmed cell death 1 ligand (PD-L1) therapy, in combination with an anti-programmed cell death 1 (PD-1) antibody, wherein the RNA is co-administered with RNA encoding an IL-12sc protein, RNA encoding an IL-15 sushi protein, and RNA encoding a GM-CSF protein.
  • PD-L1 programmed cell death 1 ligand
  • Embodiment A 5 A composition comprising RNA encoding a GM-CSF protein for use in treating a subject that has failed, or become intolerant, resistant, or refractory to an anti-programmed cell death 1 (PD-1) or anti programmed cell death 1 ligand (PD-L1) therapy, in combination with an anti-programmed cell death 1 (PD-1) antibody, wherein the RNA is co-administered with RNA encoding an IL-12sc protein, RNA encoding an IL-15 sushi protein, and RNA encoding an IFNa protein.
  • PD-1 anti-programmed cell death 1
  • P-L1 anti programmed cell death 1 ligand
  • Embodiment A 6 The composition of any one of embodiments A 1-5, wherein the
  • PD-1 anti-programmed cell death 1
  • Embodiment A 7 The composition of any one of embodiments A 1-6, wherein the
  • PD-L1 anti-programmed cell death 1 ligand
  • Embodiment A The composition of any one of embodiments A 1-7, wherein the
  • PD-1 anti-programmed cell death 1
  • PD-L1 anti programmed cell death 1 ligand
  • Embodiment A 9. The composition of any one of embodiments A 1-8, wherein the
  • PD- 1 anti -programmed cell death 1
  • PD-L1 anti -programmed cell death 1 ligand
  • Embodiment A 10. The composition of any one of embodiments A 1-9, wherein the
  • PD- 1 anti-programmed cell death 1
  • PD-L1 anti -programmed cell death 1 ligand
  • Embodiment A l l The composition of any one of embodiments A 1-10, wherein the subject has become refractory to an anti-programmed cell death 1 (PD- 1) or anti -programmed cell death 1 ligand (PD-L1) therapy.
  • PD- 1 anti-programmed cell death 1
  • PD-L1 anti -programmed cell death 1 ligand
  • Embodiment A 12 The composition of any one of embodiments A 1-11, wherein the refractory or resistant cancer is one that does not respond to a specified treatment.
  • Embodiment A 13 The composition of any one of embodiments A 1-12, wherein the refraction occurs from the very beginning of treatment.
  • Embodiment A 14 The composition of any one of embodiments A 1-13, wherein the refraction occurs during treatment.
  • Embodiment A 15 The composition of any one of embodiments A 1-14, wherein the cancer is resistant before treatment begins.
  • Embodiment A 16 The composition of any one of embodiments A 1-15, wherein the subject has a cancer that does not respond to the anti-programmed cell death 1 (PD-1) and/or anti-programmed cell death 1 ligand (PD-L1) therapy.
  • PD-1 anti-programmed cell death 1
  • PD-L1 anti-programmed cell death 1 ligand
  • Embodiment A 17 The composition of any one of embodiments A 1-16, wherein the subject has a cancer that is becoming refractory or resistant to a specified treatment.
  • Embodiment A 18 The composition of embodiment A 17, wherein the specified treatment is as an anti-PDl or anti-PD-Ll therapy.
  • Embodiment A 19 The composition of any one of embodiments A 1-18, wherein the subject has become less responsive to the therapy since first receiving it.
  • Embodiment A 20 The composition of any one of embodiments A 1-19, wherein the subject has not received the therapy, but has a type of cancer that does not typically respond to the therapy.
  • Embodiment A 21 The composition of any one of embodiments A 1-20, wherein the subject has anti-PD-1 and/or anti-PD-Ll resistant solid tumor cancer.
  • Embodiment A 22 The composition of any one of embodiments A 1-21, wherein the subject has a solid tumor cancer with acquired resistance to anti-PD-1 and/or anti-PD-Ll therapy.
  • Embodiment A 23 The composition of any one of embodiments A 1-22, wherein the subject has a solid tumor cancer with innate resistance to anti-PD-1 and/or anti-PD-Ll therapy.
  • Embodiment A 24 The composition of any one of embodiments A 1-23, wherein the subject has an advanced-stage, unresectable, or metastatic solid tumor cancer.
  • Embodiment A 25 The composition of any one of embodiments A 1-24, further processing
  • PD-1 anti-programmed cell death 1
  • PD-L1 anti-programmed cell death 1 ligand
  • Embodiment A 26 The composition of any one of embodiments A 1-25, wherein the
  • subject is human.
  • Embodiment A 27 The composition of any one of embodiments A 1-26, wherein the
  • Embodiment A 28 The composition of any one of embodiments A 1-27, wherein the
  • Embodiment A 29 The composition of any one of embodiments A 1-28, wherein the
  • subject has a cancer cell comprising a partial or total loss of beta-2 - microglobulin (B2M) function.
  • B2M beta-2 - microglobulin
  • Embodiment A 30 The composition of embodiments A 29, wherein the cancer cell has a partial loss of B2M function.
  • Embodiment A 3 E The composition embodiments A 29, wherein the cancer cell has a total loss of B2M function.
  • Embodiment A 32 The composition of any one of embodiments A 1-31, wherein the
  • partial or total loss of B2M function is assessed by comparing a cancer cell to a non-cancer cell from the same subject, optionally wherein the non-cancer cell is from the same tissue from which the cancer cell was derived.
  • Embodiment A 33 The composition of any one of embodiments A 1-32, wherein the
  • subject comprises a mutation in the B2M gene.
  • Embodiment A 34 The composition of any one of embodiments A 1-33, wherein the
  • mutation is a substitution, insertion, or deletion.
  • Embodiment A 35 The composition of any one of embodiments A 1-34, wherein the B2M gene comprises a loss of heterozygosity (LOH).
  • LHO loss of heterozygosity
  • Embodiment A 36 The composition of any one of embodiments A 1-35, wherein the
  • subject comprises a frameshift mutation.
  • Embodiment A 37 The composition of any one of embodiments A 1-36, wherein the
  • Embodiment A 38 The composition of any one of embodiments A 1-37, wherein the
  • subject comprises a frameshift mutation comprising p.LeulSfs and/or p.SerMfs.
  • Embodiment A 39 The composition of any one of embodiments A 1-38, wherein the
  • subject has a reduced level of B2M protein as compared to a subject without a partial or total loss of B2M function.
  • Embodiment A 40 The composition of any one of embodiments A 1-39, wherein the
  • MHC I histocompatibility complex class I
  • Embodiment A 4E The composition of any one of embodiments A 1-40, wherein the solid tumor cancer is an epithelial tumor, prostate tumor, ovarian tumor, renal cell tumor, gastrointestinal tract tumor, hepatic tumor, colorectal tumor, tumor with vasculature, mesothelioma tumor, pancreatic tumor, breast tumor, sarcoma tumor, lung tumor, colon tumor, melanoma tumor, small cell lung tumor, non-small cell lung cancer,
  • the solid tumor cancer is an epithelial tumor, prostate tumor, ovarian tumor, renal cell tumor, gastrointestinal tract tumor, hepatic tumor, colorectal tumor, tumor with vasculature, mesothelioma tumor, pancreatic tumor, breast tumor, sarcoma tumor, lung tumor, colon tumor, melanoma tumor, small cell lung tumor, non-small cell lung cancer,
  • neuroblastoma tumor testicular tumor, carcinoma tumor,
  • adenocarcinoma tumor adenocarcinoma tumor, seminoma tumor, retinoblastoma, cutaneous squamous cell carcinoma (CSCC), squamous cell carcinoma for the head and neck (HNSCC), head and neck cancer, osteosarcoma tumor, kidney tumor, thyroid tumor, anaplastic thyroid cancer (ATC), liver tumor, colon tumor, or other solid tumors amenable to intratumoral injection.
  • CSCC cutaneous squamous cell carcinoma
  • HNSCC squamous cell carcinoma for the head and neck
  • ATC anaplastic thyroid cancer
  • liver tumor colon tumor, or other solid tumors amenable to intratumoral injection.
  • Embodiment A 42 The composition of any one of embodiments A 1-41, wherein the solid tumor cancer is melanoma.
  • Embodiment A 43 The composition of any one of embodiments A 1-42, wherein the solid tumor cancer is not melanoma.
  • Embodiment A 44 The composition of any one of embodiments A 1-42, wherein the solid tumor cancer is melanoma, and wherein the melanoma is uveal melanoma or mucosal melanoma.
  • Embodiment A 45 The composition of any one of embodiments A 1-43, wherein the solid tumor cancer is melanoma comprising superficial, subcutaneous and/or lymph node metastases amenable for intratumoral injection.
  • Embodiment A 46 The composition of embodiment 15, wherein the solid tumor cancer is
  • HNSCC and/or mucosal melanoma with only mucosal sites are included in the HNSCC and/or mucosal melanoma with only mucosal sites.
  • Embodiment A 47 The composition of any one of embodiments A 1-46, wherein the
  • RNAs are administered as monotherapy.
  • Embodiment A 48 The composition of any one of embodiments A 1-47, wherein the
  • Embodiment A 49 The composition of any one of embodiments A 1-48, wherein at least one tumor is resistant, refractory, or intolerant to an anti -PD- 1 or anti- PD-L1 therapy and at least one tumor is not.
  • Embodiment A 50 The composition of embodiment A 49, wherein both resistant and non-resistant tumors are successfully treated.
  • Embodiment A 5 E The composition of any one of embodiments A 1-50, wherein the solid tumor cancer is stage III, subsets of stage III, stage IV, or subsets of stage IV.
  • Embodiment A 52 The composition of any one of embodiments A 1-51, wherein the solid tumor cancer is advanced-stage and unresectable.
  • Embodiment A 53 The composition of any one of embodiments A 1-52, wherein the solid tumor has spread from its origin to another site in the subject.
  • Embodiment A 54 The composition of any one of embodiments A 1-53, wherein the solid tumor cancer has one or more cutaneous or subcutaneous lesions, optionally wherein the cancer is not a skin cancer.
  • Embodiment A 55 The composition of any one of embodiments A 1-54, wherein the solid tumor cancer is stage MB, stage IIIC, or stage IV melanoma.
  • Embodiment A 56 The composition of any one of embodiments A 1-55, wherein the subject has not been treated previously with an anti -PD- 1 or anti-PD- L1 therapy.
  • Embodiment A 57 The composition of any one of embodiments A 1-56, wherein the solid tumor cancer is one in which an anti-PD-1 or anti-PD-Ll therapy is not routinely used.
  • Embodiment A 59 The composition of any one of embodiments A 1-58, wherein the subject is without other treatment options.
  • Embodiment A 60 The composition of any one of embodiments A 1-59, wherein
  • the solid tumor cancer is not melanoma, CSCC, or HNSCC;
  • an anti-PD-1 or anti-PD-Ll therapy is not routinely used; and c. there are no other suitable treatment options.
  • Embodiment A 61 The composition of any one of embodiments A 1-60, wherein the solid tumor cancer is one for which an anti -PD 1 or anti-PD-Ll therapy is routinely used, but which has not been treated with the therapy yet.
  • Embodiment A 62 The composition of any one of embodiments A 1-61, wherein the solid tumor cancer is stage MB, IIIC, or unresectable stage IV melanoma that is resistant and/or refractory to anti-PD-1 or anti-PD-Ll therapy.
  • Embodiment A 63 The composition of any one of embodiments A 1-62, wherein the solid tumor cancer comprises superficial or subcutaneous lesions and/or metastases.
  • Embodiment A 64 The composition of any one of embodiments A 1-63, wherein the subject has two or three tumor lesions.
  • Embodiment A 65 The composition of any one of embodiments A 1-64, wherein the subject has measurable disease according to the Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 criteria.
  • RECIST Response Evaluation Criteria in Solid Tumors
  • Embodiment A 66 The composition of any one of embodiments A 1-65, wherein the subject has a life expectancy of more than 3 months.
  • Embodiment A 67 The composition of any one of embodiments A 1-66, wherein the subject is at least 18 years of age.
  • Embodiment A 68 The composition of any one of the embodiments A 1-67, wherein a. the RNA encoding an IL-12sc protein comprises the nucleotide sequence of SEQ ID NO: 17 or 18, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 17 or 18; and/or b. the IL-12SC protein comprises the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 14; and/or c.
  • the RNA encoding an IL-12sc protein comprises a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the p40 portion of IL-12sc (nucleotides 1-984 of SEQ ID NO: 17 or 18) and at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the p30 portion of IL-12sc (nucleotides 1027-1623 of SEQ ID NO: 17 or 18) and further comprises nucleotides between the p40 and p35 portions encoding a linker polypeptide.
  • Embodiment A 69 The composition of any one of the embodiments A 1-68, wherein
  • the RNA encoding an IL-15 sushi protein comprises the nucleotide sequence of SEQ ID NO: 26, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 26; and/or
  • the IL-15 sushi protein comprises the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 24; and/or c.
  • the RNA encoding an IL-15 sushi protein comprises a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the sushi domain of IL-15 receptor alpha (nucleotides 1-321 of SEQ ID NO: 26) and at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to mature IL-15 (nucleotides 382-729 of SEQ ID NO: 26) and optionally further comprises nucleotides between the sushi domain of IL-15 and the mature IL- 15 encoding a linker polypeptide.
  • Embodiment A 70 The composition of any one of the embodiments A 1-69, wherein a. the RNA encoding an IFNa protein comprises the nucleotide sequence of SEQ ID NO: 22 or 23, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 22 or 23 and/or
  • the IFNa protein comprises the amino acid sequence of SEQ ID NO: 19, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 19.
  • Embodiment A 71 The composition of any one of embodiments A 1-70, wherein a. the RNA encoding a GM-CSF protein comprises the nucleotide sequence of SEQ ID NO: 29, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 29 and/or
  • the GM-CSF protein comprises the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 27.
  • Embodiment A 72 The composition of any one of embodiments A 1-71, wherein at least one RNA comprises a modified nucleoside in place of at least one uridine.
  • Embodiment A 73 The composition of any one of the preceding embodiments A 1-72, wherein at least one RNA comprises a modified nucleoside in place of each uridine.
  • Embodiment A 74 The composition of any one of embodiments A 1-73, wherein each
  • RNA comprises a modified nucleoside in place of at least one uridine.
  • Embodiment A 75 The composition of any one of embodiments A 1-74, wherein each
  • RNA comprises a modified nucleoside in place of each uridine.
  • Embodiment A 76 The composition of any one of embodiments 72-75, wherein the
  • modified nucleoside is independently selected from pseudouridine (y), N1 -methyl-pseudouridine (mV), and 5-methyl -uridine (m 5 U).
  • Embodiment A 77 The composition of any one of embodiments A 1-76, wherein at least one RNA comprises more than one type of modified nucleoside, wherein the modified nucleosides are independently selected from pseudouridine (y), N1 -methyl-pseudouridine (mV), and 5-methyl- uridine (m 5 U).
  • pseudouridine y
  • N1 -methyl-pseudouridine mV
  • m 5 U 5-methyl- uridine
  • Embodiment A 78 The composition of embodiment A 77, wherein the modified
  • nucleoside is N1 -methyl-pseudouridine (mV).
  • Embodiment A 79 The composition of any one of embodiments A 1-78, wherein at least one RNA comprises the 5’ cap im 7,3 °Gppp(mi 2 °)ApG or 3 -O-Me- m 7 G(5')ppp(5')G.
  • Embodiment A 80 The composition of any one of embodiments A 1-79, wherein each
  • RNA comprises the 5’ cap m2 7 ’ 3 °Gppp(mi 2 °)ApG or 3 -O-Me- m 7 G(5')ppp(5')G.
  • Embodiment A 81. The composition of any one of embodiments A 1-80, wherein at least one RNA comprises a 5’ UTR comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 4 and 6, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 4 and 6.
  • Embodiment A 82 The composition of any one of embodiments A 1-81, wherein each
  • RNA comprises a 5’ UTR comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 4 and 6, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 4 and 6.
  • Embodiment A 83 The composition of any one of embodiments A 1-82, wherein at least one RNA comprises a 3’ UTR comprising the nucleotide sequence of SEQ ID NO: 8, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 8.
  • Embodiment A 84 The composition of any one of embodiments A 1-83, wherein each
  • RNA comprises a 3’ UTR comprising the nucleotide sequence of SEQ ID NO: 8, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 8.
  • Embodiment A 85 The composition of any one of embodiments A 1-84, wherein at least one RNA comprises a poly- A tail.
  • Embodiment A 86 The composition of any one of embodiments A 1-85, wherein each
  • RNA comprises a poly- A tail.
  • Embodiment A 87 The composition of embodiment A 84 or A 85, wherein the poly-A tail comprises at least 100 nucleotides.
  • Embodiment A 88 The composition of any one of embodiments A 85-87, wherein the poly-A tail comprises the poly-A tail shown in SEQ ID NO: 30.
  • Embodiment A 89 The composition of any one of embodiments A 1-88, wherein one or more RNA comprises:
  • a 5’ cap comprising m2 7 ’ 3 °Gppp(mi 2 °)ApG or 3'-0-Me-m 7 G(5')ppp(5')G;
  • a 5’ UTR comprising (i) a nucleotide sequence selected from the group consisting of SEQ ID NOs: 4 and 6, or (ii) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 4 and 6;
  • a 3’ UTR comprising (i) the nucleotide sequence of SEQ ID NO: 8, or (ii) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:8; and d. a poly-A tail comprising at least 100 nucleotides.
  • Embodiment A 90 The composition of embodiment A 89, wherein the poly-A tail
  • Embodiment A 91 The composition of any one of embodiments A 1-90, wherein the
  • composition is used in treating an advanced-stage, unresectable, or metastatic solid tumor cancer in a human.
  • Embodiment A 92 The composition of any one of embodiments A 1-91, wherein treating the solid tumor comprises reducing the size of a tumor or preventing cancer metastasis in a subject.
  • Embodiment A 93 The composition of any one of embodiments A 1-92, wherein the
  • RNAs are administered at the same time.
  • Embodiment A 94 The composition of any one of embodiments A 1-93, wherein the
  • RNAs are administered via injection.
  • Embodiment A 95 The composition of embodiments A 93 or A 94, wherein the RNAs are mixed together in liquid solution prior to injection.
  • Embodiment A 96 The composition of any one of embodiments A1 - A96, wherein the solid tumor cancer comprises lymphoma.
  • Embodiment A 97 The composition of any one of embodiments A1 - A96, wherein the solid tumor cancer comprises Hodgkin lymphoma.
  • Embodiment A 98 The composition of any one of embodiments A1 - A96, wherein the solid tumor cancer comprises Non-Hodgkin lymphoma.
  • Embodiment B l A method for treating an advanced-stage, unresectable, or metastatic solid tumor cancer comprising administering to a subject having advanced-stage, unresectable, or metastatic solid tumor cancer i. RNA encoding an IL-12sc protein, RNA encoding an IL-15 sushi protein, RNA encoding an IFNa protein, and RNA encoding a GM-CSF protein; and
  • PD-1 anti-programmed cell death 1
  • Embodiment B 2. The method of embodiment B 1, wherein the solid tumor cancer is stage III, subsets of stage III, stage IV, or subsets of stage IV.
  • Embodiment B 3. The method of any one of the preceding embodiments, wherein the solid tumor cancer is advanced-stage and unresectable.
  • Embodiment B 4. The method of any one of the preceding embodiments, wherein the solid tumor has spread from its origin to another site in the subject.
  • Embodiment B 5 The method of any one of the preceding embodiments, wherein the solid tumor cancer is stage III, stage MB, stage IIIC, or stage IV cancer.
  • Embodiment B 6 The method of embodiment B5, wherein the stage IV cancer is
  • Embodiment B 7 The method of any one of the preceding embodiments, wherein the solid tumor cancer is melanoma, cutaneous squamous cell cancer (CSCC), squamous cell carcinoma for the head and neck (HNSCC), non-small cell lung cancer, kidney cancer, head and neck cancer, thyroid cancer, colon cancer, liver cancer, ovarian cancer, breast cancer or other solid tumors amenable to intratumoral injection.
  • CSCC cutaneous squamous cell cancer
  • HNSCC squamous cell carcinoma for the head and neck
  • non-small cell lung cancer kidney cancer, head and neck cancer
  • thyroid cancer colon cancer
  • liver cancer ovarian cancer
  • breast cancer breast cancer or other solid tumors amenable to intratumoral injection.
  • Embodiment B 8. The method of any one of the preceding embodiments, wherein the solid tumor cancer is melanoma.
  • Embodiment B 9 The method of any one of the preceding embodiments, wherein the solid tumor cancer is breast cancer, e.g., breast sarcoma, triple negative breast cancer.
  • breast cancer e.g., breast sarcoma, triple negative breast cancer.
  • Embodiment B 10 The method of any one of the preceding embodiments, wherein the solid tumor cancer is ovarian cancer.
  • Embodiment B 11 The method of embodiment BIO, wherein the ovarian cancer is
  • Embodiment B 12 The method of any one of the preceding embodiments, wherein the solid tumor cancer is thyroid cancer.
  • Embodiment B 13 The method of embodiment B 12, wherein the thyroid cancer is
  • ATC anaplastic thyroid cancer
  • Embodiment B 14 The method of any one of the preceding embodiments, wherein the solid tumor cancer has one or more cutaneous or subcutaneous lesions (e.g., metastasis), but is not a skin cancer.
  • the solid tumor cancer has one or more cutaneous or subcutaneous lesions (e.g., metastasis), but is not a skin cancer.
  • Embodiment B 15 The method of any one of the preceding embodiments, wherein the solid tumor cancer is stage IIIB, stage IIIC, or stage IV melanoma.
  • Embodiment B 16 The method of any one of the preceding embodiments, wherein the subject has failed, or become intolerant, resistant, or refractory to an anti-programmed cell death 1 (PD-1) or anti-programmed cell death 1 ligand (PD-L1) therapy.
  • PD-1 anti-programmed cell death 1
  • PD-L1 anti-programmed cell death 1 ligand
  • Embodiment B 17 The method of any one of the preceding embodiments, wherein the solid tumor cancer is melanoma, non-small cell lung cancer, kidney cancer, head and neck cancer.
  • Embodiment B 18. The method of any one of the preceding embodiments, wherein the subject has not been treated previously with an anti -PD-1 or anti-PD- L1 therapy.
  • Embodiment B 19 The method of any one of the preceding embodiments, wherein the solid tumor cancer is one in which an anti-PD-1 or anti-PD-Ll therapy is not routinely used.
  • Embodiment B 20 The method of embodiment B 19, wherein the solid tumor cancer is not melanoma, non-small cell lung cancer, kidney cancer, head and neck cancer, cutaneous squamous cell carcinoma (CSCC), head and neck squamous cell carcinoma (HNSCC).
  • the solid tumor cancer is not melanoma, non-small cell lung cancer, kidney cancer, head and neck cancer, cutaneous squamous cell carcinoma (CSCC), head and neck squamous cell carcinoma (HNSCC).
  • CSCC cutaneous squamous cell carcinoma
  • HNSCC head and neck squamous cell carcinoma
  • Embodiment B 21 The method of any one of the preceding embodiments, wherein the subject is without other treatment options.
  • Embodiment B 22 The method of embodiment Bl, wherein
  • the solid tumor cancer is not melanoma, cutaneous squamous cell carcinoma, or head and neck squamous cell carcinoma;
  • an anti-PD-1 or anti-PD-Ll therapy is not routinely used.
  • Embodiment B 23 The method of any one of the preceding embodiments, wherein the solid tumor cancer is one for which an anti-PDl or anti-PD-Ll therapy is routinely used, but which has not been treated with the therapy yet
  • HNSCC head and neck cancer
  • CSCC CSCC
  • Embodiment B 24 The method of embodiment Bl, wherein the solid tumor cancer is stage IIIB, IIIC, or unresectable stage IV melanoma that is resistant and/or refractory to anti-PD-1 or anti-PD-Ll therapy.
  • Embodiment B 25 The method of embodiment Bl, wherein the solid tumor cancer is stage III or unresectable stage IV of cutaneous squamous cell carcinoma or head and neck squamous cell carcinoma that is resistant and/or refractory to anti-PD-1 or anti-PD-Ll therapy.
  • Embodiment B 26 The method of any one of the preceding embodiments, wherein the solid tumor cancer comprises superficial or subcutaneous lesions and/or metastases.
  • Embodiment B 27 The method of any one of the preceding embodiments, wherein the solid tumor cancer is an epithelial tumor, prostate tumor, ovarian tumor, renal cell tumor, gastrointestinal tract tumor, hepatic tumor, colorectal tumor, tumor with vasculature, mesothelioma tumor, pancreatic tumor, breast tumor, sarcoma tumor, lung tumor, colon tumor, melanoma, small cell lung tumor, neuroblastoma tumor, testicular tumor, carcinoma tumor, adenocarcinoma tumor, seminoma tumor, retinoblastoma, cutaneous squamous cell carcinoma (CSCC), squamous cell carcinoma for the head and neck (HNSCC), head and neck cancer, or osteosarcoma tumor.
  • the solid tumor cancer is an epithelial tumor, prostate tumor, ovarian tumor, renal cell tumor, gastrointestinal tract tumor, hepatic tumor, colorectal tumor, tumor with vasculature, mesothelioma tumor,
  • Embodiment B 28 The method of any one of the preceding embodiments, wherein the subject has two or three tumor lesions.
  • Embodiment B 29 The method of any one of the preceding embodiments, wherein the subject has measurable disease according to the Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 criteria.
  • RECIST Response Evaluation Criteria in Solid Tumors
  • Embodiment B 30 The method of any one of the preceding embodiments, wherein the subject has a life expectancy of more than 3 months.
  • Embodiment B 3 E The method of any one of the preceding embodiments, wherein the subject is at least 18 years of age.
  • Embodiment B 32 A method for treating an advanced-stage melanoma, cutaneous
  • squamous cell carcinoma or head and neck squamous cell carcinoma, comprising administering to a subject having an advanced-stage melanoma,
  • RNA encoding an IL-12sc protein RNA encoding an IL-15 sushi protein, RNA encoding an IFNa protein, and RNA encoding a GM-CSF protein;
  • the subject is at least 18 years of age
  • the subject has failed prior anti -PD 1 or anti-PD-Ll therapies; c. the subject has a minimum of 2 lesions; and d. the melanoma comprises a tumor that is suitable for direct
  • Embodiment B 33 The method of embodiment B 32, wherein the subject has measurable disease according to the Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 criteria.
  • RECIST Response Evaluation Criteria in Solid Tumors
  • Embodiment B 34 The method of embodiment B 32, wherein the subject has a life
  • Embodiment B 35 The method of any one of the preceding embodiments, wherein the anti -PD 1 antibody is cemiplimab, pembrolizumab, nivolumab, MEDI0608, PDR001, PF-06801591, BGB-A317, pidilizumab, TSR- 042, AGEN-2034, A-0001, BGB-108, BI-754091, CBT-501, ENUM- 003, ENUM-388D4, IBI-308, JNJ-63723283, JS-001, JTX-4014, JY- 034, CLA-134, STIA-1110, 244C8, or 388D4.
  • the anti -PD 1 antibody is cemiplimab, pembrolizumab, nivolumab, MEDI0608, PDR001, PF-06801591, BGB-A317, pidilizumab, TSR- 042, AGEN-2034, A-0001, BGB
  • Embodiment B 36 The method of any one of the preceding embodiments, wherein the anti-PDl antibody is cemiplimab.
  • Embodiment B 37 The method of any one of the preceding embodiments, wherein the anti-PDl antibody is administered at a dose of about 0.1-600 mg.
  • Embodiment B 38 The method of any one of the preceding embodiments, wherein the anti-PDl antibody is administered at a dose of 200 mg, 240 mg, or 350 mg.
  • Embodiment B 39 The method of any one of the preceding embodiments, wherein the anti-PDl antibody is administered via injection.
  • Embodiment B 40 The method of any one of the preceding embodiments, wherein the anti -PD 1 antibody is administered intravenously.
  • Embodiment B 4E The method of any one of the preceding embodiments, wherein the anti-PD-1 antibody is administered once every three weeks.
  • Embodiment B 42 The method of any one of the preceding embodiments, wherein the
  • RNAs are injected intratum orally.
  • Embodiment B 43 The method of any one of the preceding embodiments, wherein the
  • RNAs and the anti-PD-1 antibody are administered for about 8 months.
  • Embodiment B 44 The method of any one of the preceding claims, wherein the RNAs are administered once every week.
  • Embodiment B 45 The method of any one of the preceding embodiments, wherein the
  • RNAs are administered for a maximum of 52 weeks.
  • Embodiment B 46 The method of any one of the preceding embodiments, wherein the
  • IFNa protein is an IFNa2b protein.
  • Embodiment B 47 The method of any one of the preceding embodiments, wherein
  • the RNA encoding an IL-12sc protein comprises the nucleotide sequence of SEQ ID NO: 17 or 18, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 17 or 18; and/or b.
  • the IL-12SC protein comprises the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 14; and/or
  • RNA encoding an IL-12sc protein comprises a nucleotide
  • nucleotides 1027-1623 of SEQ ID NO: 17 or 18 further comprises nucleotides between the p40 and p35 portions encoding a linker polypeptide.
  • Embodiment B 48 The method of any one of the preceding embodiments, wherein
  • the RNA encoding an IL-15 sushi protein comprises the nucleotide sequence of SEQ ID NO: 26, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 26; and/or
  • the IL-15 sushi protein comprises the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 24; and/or
  • the RNA encoding an IL-15 sushi protein comprises a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the sushi domain of IL-15 receptor alpha
  • IL-15 97%, 96%, 95%, 90%, 85%, or 80% identity to mature IL-15 (nucleotides 382-729 of SEQ ID NO: 26) and optionally further comprises nucleotides between the sushi domain of IL-15 and the mature IL-15 encoding a linker polypeptide.
  • Embodiment B 49 The method of any one of the preceding embodiments, wherein
  • RNA encoding an IFNa protein comprises the nucleotide
  • the IFNa protein comprises the amino acid sequence of SEQ ID NO: 19, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 19.
  • Embodiment B 50 The method of any one of the preceding embodiments, wherein
  • the RNA encoding a GM-CSF protein comprises the nucleotide sequence of SEQ ID NO: 29, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 29 and/or
  • the GM-CSF protein comprises the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 27.
  • Embodiment B 52 The method of any one of the preceding embodiments, wherein at least one RNA comprises a modified nucleoside in place of each uridine.
  • Embodiment B 53 The method of any one of the preceding embodiments, wherein each
  • RNA comprises a modified nucleoside in place of at least one uridine.
  • Embodiment B 54 The method of any one of the preceding embodiments, wherein each
  • RNA comprises a modified nucleoside in place of each uridine.
  • Embodiment B 55 The method of any one of embodiments B 51-54, wherein the modified nucleoside is independently selected from pseudouridine (y), Nl- methyl-pseudouridine (ih ⁇ y), and 5-methyl-uridine (m5U).
  • Embodiment B 56 The method of any one of the preceding embodiments, wherein at least one RNA comprises more than one type of modified nucleoside, wherein the modified nucleosides are independently selected from pseudouridine (y), N1 -methyl-pseudouridine (m 1 y), and 5-methyl- uridine (m5U).
  • pseudouridine y
  • N1 -methyl-pseudouridine m 1 y
  • m5U 5-methyl- uridine
  • Embodiment B 57 The method of embodiment B 56, wherein the modified nucleoside is
  • Embodiment B 58 The method of any one of the preceding embodiments, wherein at least one RNA comprises the 5’ cap m27,3’-0Gppp(ml2’-0)ApG or 3 -0- Me-m7G(5')ppp(5')G.
  • Embodiment B 59 The method of any one of the preceding embodiments, wherein each
  • RNA comprises the 5’ cap m27,3’-0Gppp(ml2’-0)ApG or 3 -O-Me- m7G(5')ppp(5')G.
  • Embodiment B 60 The method of any one of the preceding embodiments, wherein at least one RNA comprises a 5’ UTR comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 4 and 6, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 4 and 6.
  • Embodiment B 61 The method of any one of the preceding embodiments, wherein each
  • RNA comprises a 5’ UTR comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 4 and 6, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or
  • Embodiment B 62 The method of any one of the preceding embodiments, wherein at least one RNA comprises a 3’ UTR comprising the nucleotide sequence of SEQ ID NO: 8, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 8.
  • Embodiment B 63 The method of any one of the preceding embodiments, wherein each
  • RNA comprises a 3’ UTR comprising the nucleotide sequence of SEQ ID NO: 8, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 8.
  • Embodiment B 64 The method of any one of the preceding embodiments, wherein at least one RNA comprises a poly-A tail.
  • Embodiment B 65 The method of any one of the preceding embodiments, wherein each
  • RNA comprises a poly-A tail.
  • Embodiment B 66 The method of embodiment B 64 or 65, wherein the poly-A tail
  • Embodiment B 67 The method of embodiment B 64 or 65, wherein the poly-A tail
  • Embodiment B 68 The method of any one of the preceding embodiments, wherein one or more RNA comprises:
  • a 5’ cap comprising m27,3’-0Gppp(ml2’-0)ApG or 3'-0-Me- m7G(5')ppp(5')G;
  • a 5’ UTR comprising (i) a nucleotide sequence selected from the group consisting of SEQ ID NOs: 4 and 6, or (ii) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 4 and 6;
  • a 3’ UTR comprising (i) the nucleotide sequence of SEQ ID NO: 8, or (ii) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:8; and d. a poly-A tail comprising at least 100 nucleotides.
  • Embodiment B 69 The method of embodiment B 68, wherein the poly-A tail comprises
  • Embodiment B 70 The method of any one of the preceding embodiments, wherein the subject is human.
  • Embodiment B 7E The method of any one of the preceding embodiments, wherein
  • treating the solid tumor comprises reducing the size of a tumor or preventing cancer metastasis in a subject.
  • Embodiment B 72 The method of any one of the preceding embodiments, wherein the
  • RNAs are administered at the same time.
  • Embodiment B 73 The method of any one of the preceding embodiments, wherein the
  • RNAs are administered via injection.
  • Embodiment B 74 The method of embodiment B 72 or 73, wherein the RNAs are mixed together in liquid solution prior to injection.
  • Fig. 1A shows an exemplary overall design of treatments.
  • Fig. IB shows an exemplary treatment schedule for administration of the cytokine RNA mixture as monotherapy.
  • Fig 1C shows an exemplary treatment schedule for administration of the cytokine RNA mixture as combination therapy with an anti -PD- 1 antibody.
  • Figs. 2A - 21 show the creation and characterization of a murine model of acquired resistance to anti-PD-1 therapy.
  • Figs. 2A - 2B show the generation of a PD-1 resistant tumor line.
  • Fig. 2A is a diagram of in vivo passaging approach. Briefly, C57BL6 mice bearing MC38 tumors were treated with anti-PD-1 antibody (clone RMP1-14), growing tumors were excised, and cells from the tumors were cultured ex vivo prior to implantation into naive mice.
  • Fig. 2B shows tumor growth curves for MC38 and MC38-resistant tumor cell lines implanted in C57BL6/J mice treated with 10 mg/kg anti-PD-1 antibody
  • Figs. 2C - 2E show that MC38-resistant cells do not exhibit known molecular mechanisms of PD-1 resistance. MC38 and MC38-resistant cells were cultured in vitro and expression of different proteins was assayed by flow cytometry.
  • Fig. 2C is a series of graphs showing surface expression of PD-L1, B2M and IFNGR1 and IFNGR2. Line, unstained; filled, stained sample.
  • Fig. 2D is a graph showing PD-L1 expression following IFNy treatment in vitro.
  • Fig. 2E is a graph showing expression of SIINFEKL-MHC I complex in OVA-transduced cells.
  • Figs. 2F-2I show subcutaneous tumors excised and profiled by RNA-sequencing.
  • Fig. 2G shows expression of IFNy target genes is reduced in MC38-resistant tumors.
  • Fig. 2H shows MCPCounter analysis estimating relative immune abundance, revealing significantly reduced T, NK, B cell lineage and monocytic lineage cells. *, p ⁇ 0.05.
  • Fig. 21 shows immune infiltration by flow cytometry in CD8 + T cells (CD45 + CD3 + CD4 CD8 +) , CD4 + T cells (CD45 + CD3 + CD4 + CD8 ), macrophages
  • Fig. 3 shows that MC38-resistant cells do not express PD-L2.
  • MC38-resistant cells were cultured in vitro and expression of different proteins was assayed by flow cytometry. PD-L2 expression following IFNy treatment is shown.
  • Figs. 4A -4B show reduced frequency of immune cells in resistant tumors by immunohistochemical staining. Paraffin embedded MC38 and MC38-resistant tumors were analyzed by immunohistochemical staining for infiltration of CD45 + cells (dark color)
  • Fig. 4A shows representative images.
  • Fig. 4B shows quantification.
  • Figs. 5A-5B show reduced immunogenicity of resistant tumors.
  • Cytotoxic T lymphocyte (CTL) cultures were generated from 5 individual C57BL6 mice bearing parental MC38 tumors that exhibited complete regression in response to PD-1 blockade. CTLs were co-cultured with MC38 and resistant tumor cells, and killing (Fig. 4A) and IFNy release (Fig. 5B) were measured.
  • CTL Cytotoxic T lymphocyte
  • FIGs. 6A - 6D show that C57BL6/J mice bearing subcutaneous MC38 or
  • MC38-resistant tumors were successfully treated with intratumoral injection of cytokine RNA mixture (Figs. 6B and 6D) as measured by tumor burden. mRNA treatments were administered every four days (as indicated by arrows) at a dose of 40 pg total mRNA.“Luc” (Figs. 6A and 6C) indicates luciferase control mRNA.
  • FIG. 7 shows that C57BL6/J mice bearing subcutaneous MC38 or MC38- resistant tumors were successfully treated with intratumoral injection of cytokine RNA mixture as measured by overall survival. mRNA treatments were administered every four days (as indicated by arrows) at a dose of 40 pg total mRNA.“Luc” indicates luciferase control mRNA.
  • Figs. 8A - 8B shows flow cytometry analysis of beta-2 microglobulin (B2M) surface expression in MC38 (Fig. 8A) and MC38 with deletion of B2M (Fig. 8B).
  • B2M beta-2 microglobulin
  • Figs. 9A - 9D show that a combination of the cytokine RNA mixture with anti -PD- 1 antibody enhanced survival in a dual flank B16F 10 cancer model (Fig. 9A) and MC38 tumor model (Fig. 9B). Overall survival in single flank MC38-B2M knockout treated with cytokine RNA mixture (Fig. 9C) or a heterologous dual flank model with MC38-B2M knockout/MC38-WT tumors (Fig. 9D).
  • Fig. 10 shows changes in tumor volume after cytokine mRNA mixture, anti-
  • PD-1 or a combination of cytokine mRNA mixture and anti -PD- 1 therapy in various in vivo solid tumor cancer models.
  • Numerical values correspond to tumor volume changes from baseline (AT/AC, %). Changes in tumor volume for each treated (T) and vehicle control (C) group are calculated for each animal by subtracting the tumor volume on the day of first treatment from the tumor volume on the last day when all the control mice were still alive. The median AT is calculated for the treated group, and the median AC is calculated for the vehicle control group. The ratio AT/AC is calculated and expressed as percentage.
  • Fig. 11 shows a“peri-tumorally,” or“peri -tumoral,” area that is about 2-mm wide and is adjacent to the invasive front of the tumor periphery.
  • the peri -tumoral area comprises host tissue.
  • Table 1 provides a listing of certain sequences referenced herein.
  • a“cytokine RNA mixture,” also sometimes referred to as“cytokine mRNA mixture,”“mRNA cytokine mixture,” or“RNA cytokine mixture” comprises RNA encoding IFNa, RNA encoding IL-15 sushi, RNA encoding IL-12sc, and RNA encoding GM- CSF, as described herein.
  • “PD-1” may also be referred to as“programmed cell death 1” or“programmed cell death-1.”
  • “PD-L1” may also be referred to as“programmed cell death 1 ligand,”“programmed cell death-1 ligand 1,” or“programmed cell death-ligand 1.”
  • an“advanced stage solid tumor cancer,” sometimes referred to herein as “advanced solid tumor,” or“advanced solid tumor cancer,” comprises a solid tumor cancer whose stage is identified as stage III, subsets of stage III, stage IV, or subsets of stage IV, assessed by a known system, e.g ., the tumor, node, and metastasis (TNM) staging system developed by the American Joint Committee on Cancer (AJCC) (see AJCC Cancer Staging Manual, 8 th Edition).
  • the TNM staging system is used for solid tumor cancers other than melanoma.
  • the cancer is melanoma or advanced melanoma, which comprises stage IIIB, stage IIIC, or stage V as assessed by the AJCC melanoma staging (edition 8, 2018).
  • AJCC melanoma staging are provided in Gershenwald JE, Scolyer RA, Hess KR, et al. Melanoma of the skin.
  • Amin MB ed. AJCC Cancer Staging Manual. 8th ed. Chicago, ILAJCC-Springer; 2017:563- 585, the entire contents of which are incorporated herein by reference.
  • the cancer is cutaneous squamous cell carcinoma (CSCC), or squamous cell carcinoma of the head and neck (HNSCC), both of which may be advanced.
  • CSCC cutaneous squamous cell carcinoma
  • HNSCC squamous cell carcinoma of the head and neck
  • “Tumor” may also be referred to herein as“neoplasm”.
  • “neoplasm” the terms“solid tumor” and“solid neoplasm” are interchangeable.
  • An“unresectable” (e.g, advanced- stage unresectable) cancer typically cannot be removed with surgery.
  • RECIST Response Evaluation Criteria for Solid Tumours (also Tumors) provides a methodology to evaluate the activity and efficacy of cancer therapeutics in solid tumors.
  • RECIST guidelines were created by the RECIST Working Group comprising representatives from the European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States and Canadian Cancer Trials Group, as well as several
  • nodes (whether target or non-target) must have reduction in short axis to ⁇ 10 mm.
  • Partial Response At least a 30% decrease in the sum of diameters of target lesions, taking as reference the baseline sum diameters.
  • PD Progressive Disease
  • SD Stable Disease
  • Section 4.3.3 of the guidelines (page 233 of Eisenhauer) provides the following regarding evaluation of non-target lesions:
  • non-target lesions may actually be measurable, they need not be measured and instead should be assessed only qualitatively at the time points specified in the protocol.
  • CR Complete Response
  • PD Progressive Disease
  • a subject having“innate” or“primary” resistance to an anti-PD-1 or anti-PD-Ll therapy does not initially respond to anti-PD-1 or anti-PD-Ll therapy.
  • a subject having innate or primary resistance never demonstrated a clinical response to PD-1/PD-L1 blockade. See, e.g. , Sharma et al. (2017) Cell 168:707-723 at 709; see also , Hugo et al. (2016) Cell 165 (1) 35-44; see also , Nowicki et al. (2016) Cancer J. 24(1): 47-53, the entire contents of which are incorporated herein by reference.
  • a subject with innate resistance to an anti-PD-1 or anti-PD-Ll therapy is characterized after treatment with anti-PD-1 or anti-PD-Ll therapy (any length of time) as having Progressive Disease or Stable Disease according to RECIST criteria (version 1.1).
  • a subject with innate resistance to an anti-PD-1 or anti-PD-Ll therapy is characterized after treatment with anti-PD-1 or anti-PD-Ll therapy (any length of time) as having non-CR/Non-PD for non-target lesions comprising viable cancer cells.
  • a subject with innate resistance to an anti-PD-1 therapy is characterized after treatment with anti-PD-1 therapy (any length of time) as having Progressive Disease according to RECIST criteria (version 1.1).
  • a subject with innate resistance to an anti-PD-Ll therapy is characterized after treatment with anti-PD-Ll therapy (any length of time) as having Progressive Disease according to RECIST criteria (version 1.1).
  • a subject with innate resistance to an anti-PD-1 therapy is characterized after treatment with anti-PD-1 therapy (any length of time) as having Stable Disease according to RECIST criteria (version 1.1).
  • a subject with innate resistance to an anti-PD-Ll therapy is characterized after treatment with anti-PD-Ll therapy (any length of time) as having Stable Disease according to RECIST criteria (version 1.1).
  • a subject with innate resistance to an anti-PD-1 or anti-PD-Ll therapy is characterized after treatment with anti-PD-1 or anti-PD-Ll therapy (any length of time) as having at least a 20% increase in the longest diameter of a solid tumor and/or the appearance of one or more new solid tumors.
  • a subject with innate resistance to an anti- PD-1 is characterized after treatment with anti-PD-1 therapy (any length of time) as having at least a 20% increase in the longest diameter of solid tumors and/or the appearance of one or more new solid tumors.
  • a subject with innate resistance to an anti-PD- L1 therapy is characterized after treatment with anti-PD-Ll therapy (any length of time) as having at least a 20% increase in the longest diameter of solid tumors and/or the appearance of one or more new solid tumors.
  • the increase in the longest diameter is an increase of at least 5 mm.
  • the length of time is about 6 weeks, about 8 weeks, or at least 6 or 8 weeks. In some embodiments, the length of time is 2, 3, 6, 12, or more months.
  • the solid tumor is a primary tumor.
  • the solid tumor is an injectable tumor.
  • the solid tumor has been injected with the cytokine mRNA mixture.
  • the solid tumor has been selected for injection with the cytokine mRNA mixture.
  • the solid tumor is a subcutaneous lesion >0.5 cm in longest diameter.
  • the solid tumor is within a group of multiple injectable merging lesions that are confluent.
  • the solid tumor is within a group of multiple injectable merging lesions that are confluent and have the longest diameter (sum of diameters of all involved target lesions) of >0.5 cm.
  • the solid tumor is not bleeding or weeping.
  • the longest diameter of the solid tumor is at least 10 mm (e.g ., as measured by Computed Tomography (CT) scan or caliper).
  • the solid tumor is in the chest of a subject and longest diameter of the solid tumor is at least 20 mm (e.g., as measured by chest X-ray). In some embodiments, the solid tumor is in a lymph node. In some embodiments, the lymph node is at least 15 mm in short axis (e.g, when assessed by CT scan). In some embodiments, the solid tumor is a lymphoma. In some embodiments, a subject with innate resistance to an anti- PD-1 or anti-PD-Ll therapy is characterized after treatment with anti-PD-1 or anti-PD-Ll therapy (any length of time) as having no response or stable disease according to the Lugano Classification.
  • a subject with innate resistance to an anti-PD-1 or anti-PD-Ll therapy is characterized after treatment with anti-PD-1 or anti-PD-Ll therapy (any length of time) as having progressive disease according to the Lugano Classification.
  • a subject with innate resistance to an anti-PD-1 or anti-PD-Ll therapy is characterized after treatment with anti-PD-1 or anti-PD-Ll therapy (any length of time) as having a lymphoma tumor within a lymph node.
  • a subject with innate resistance to an anti-PD-1 or anti-PD-Ll therapy is characterized after treatment with anti-PD-1 or anti-PD-Ll therapy (any length of time) as having a lymphoma tumor within a lymph node, wherein the lymph node has (i) a longest diameter of greater than 1.5 cm, and (ii) an increase of at least 50% from the product of the perpendicular diameters (PPDs) nadir.
  • PPDs perpendicular diameters
  • the increase in the longest diameter is an increase of at least 5 mm.
  • the length of time is about 6 weeks, about 8 weeks, or at least 6 or 8 weeks. In some embodiments, the length of time is 2, 3, 6, 12, or more months.
  • a subject having“acquired” or“adaptive” resistance to an anti-PD-1 or anti-PD-Ll therapy initially responds to therapy (e.g any level of response), but after a period of time relapses and progresses.
  • response to therapy is assessed as per RECIST criteria (version 1.1).
  • acquired or adaptive resistance to an anti-PD-1 or anti-PD-Ll therapy is seen in subjects who eventually progresses while on therapy despite an initial Complete Response or Partial Response, all according to RECIST criteria (version 1.1).
  • acquired or adaptive resistance to an anti-PD-1 or anti-PD-Ll therapy is seen in subjects who are unresponsive to re-initiation of an anti-PD-1 or anti-PD-Ll therapy. See, Sharma et al. (2017) Cell 168:707-723 at 708; see also , Nowicki et al. (2016) Cancer J. 24(1): 47-53, the entire contents of which are incorporated herein by reference.
  • a subject with adaptive resistance to an anti-PD-1 therapy comprises a solid tumor whose volume (i) decreased for a period of time after anti-PD-1 therapy began; and then (ii) increased after the period of time despite continued anti-PD-1 therapy.
  • a subject with adaptive resistance to an anti-PD-Ll therapy comprises a solid tumor whose volume (i) decreased for a period of time after anti-PD-Ll therapy began; and then (ii) increased after the period of time despite continued anti-PD-Ll therapy.
  • the adaptive resistance is associated with an acquired underlying mechanism of resistance.
  • the adaptive resistance is associated with a mutation or an epigenetic change.
  • the adaptive resistance is associated with a mutation in a B2M gene.
  • the period of time is from 6 to 12 months. In some embodiments, the period of time is from 6 to 18 months. In some embodiments, the period of time is from 6 to 36 months.
  • the period of time is from 3 to 9 months. In some embodiments, the period of time is from 3 to 24 months. In some embodiments, the period of time is from 12 to 24 months. In some embodiments, the period of time is at least about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, or about 24 months. In some embodiments, the period of time is at least about 4 months. In some embodiments, the period of time is at least about 6 months. In some embodiments, the period of time is at least about 12 months. In some embodiments, the period of time is at least about 24 months. In some embodiments, the period of time is at least about 30 months.
  • the period of time is at least about 36 months.
  • a subject with adaptive resistance to an anti -PD- 1 or anti-PD-Ll therapy was characterized at any point during treatment as having a Complete Response and thereafter (and during treatment) was characterized as having Progressive Disease according to RECIST criteria (version 1.1).
  • a subject with adaptive resistance to an anti -PD- 1 or anti-PD-Ll therapy was characterized at any point during treatment as having a Partial Response and thereafter (and during treatment) was characterized as having a Progressive Disease or Stable Disease, all according to RECIST criteria (version 1.1).
  • a subject with adaptive resistance to an anti-PD-1 or anti-PD-Ll therapy was characterized at any point during treatment as having a Partial Response and thereafter (and during treatment) was characterized as having Progressive Disease according to RECIST criteria (version 1.1).
  • RECIST criteria version 1.1
  • a subject with adaptive resistance to an anti-PD-1 or anti-PD-Ll therapy was characterized at any point during treatment as having a Partial Response and thereafter (and during treatment) was characterized as having Stable Disease according to RECIST criteria (version 1.1).
  • the longest diameter of solid tumors in the subject decreased by at least 30% after the anti-PD-1 or anti-PD-Ll therapy began and then increased.
  • the longest diameter of solid tumors in the subject decreased by at least 30% after the anti-PD-1 or anti-PD-Ll therapy began and then increased by at least 20%. In some embodiments, the longest diameter of solid tumors in the subject decreased by at least 30% after the anti-PD-1 or anti-PD-Ll therapy began and then one or more new solid tumors appeared. In some embodiments, a subject with adaptive resistance to an anti-PD-1 or anti-PD- Ll therapy was characterized at any point during treatment as having least a 30% decrease in the longest diameter of solid tumors and thereafter (and during treatment) was characterized as having at least a 20% increase in the longest diameter of a solid tumors and/or the appearance of one or more new solid tumors.
  • the increase in the longest diameter is an increase of at least 5 mm.
  • a subject with adaptive resistance to an anti- PD-1 or anti-PD-Ll therapy was characterized at any point during treatment as having a disappearance of a solid tumor (e.g., every solid tumor that was present if more than one solid tumor was present) and thereafter (and during treatment) was characterized as having the reappearance of the solid tumor (e.g., in the same location as a solid tumor that disappeared) and/or the appearance of one or more new solid tumors.
  • the solid tumor is a primary tumor.
  • the solid tumor is an injectable tumor.
  • the tumor has been injected with the cytokine mRNA mixture.
  • the tumor has been selected for injection with the cytokine mRNA mixture.
  • the solid tumor is a subcutaneous lesion >0.5 cm in longest diameter.
  • the solid tumor is within a group of multiple injectable merging lesions that are confluent.
  • the solid tumor is within a group of multiple injectable merging lesions that are confluent and have the longest diameter (sum of diameters of all involved target lesions) of >0.5 cm.
  • the solid tumor is not bleeding or weeping.
  • the longest diameter of the solid tumor is at least 10 mm (e.g., as measured by Computed Tomography (CT) scan or caliper).
  • CT Computed Tomography
  • the solid tumor is in the chest of a subject and longest diameter of the solid tumor is at least 20 mm (e.g, as measured by chest X-ray). In some embodiments, the solid tumor is in a lymph node. In some embodiments, the lymph node is at least 15 mm in short axis (e.g, when assessed by CT scan). In some embodiments, the solid tumor is a lymphoma. In some embodiments, a subject with adaptive resistance to an anti-PD-1 or anti-PD-Ll therapy was characterized at any point during treatment as having a complete response and thereafter (and during treatment) was characterized as having progressive disease according to the Lugano Classification.
  • a subject with adaptive resistance to an anti-PD-1 or anti-PD-Ll therapy was characterized at any point during treatment as having at least a 50% decrease in the sum of the product of the perpendicular diameters (PPDs) for multiple lesions (e.g. for 1, 2, 3, 4, 5, or 6 lymph node or extranodal sites) and thereafter (and during treatment) was characterized as having a lymphoma tumor within a lymph node, wherein the lymph node has (i) a longest diameter of greater than 1.5 cm, and (ii) an increase of at least 50% from the PPD nadir.
  • PPDs perpendicular diameters
  • A“refractory” or“resistant” cancer is one that does not respond to a specified treatment. In some embodiments, refraction occurs from the very beginning of treatment. In some embodiments, refraction occurs during treatment. In some embodiments, a cancer is resistant before treatment begins. In some embodiments, a cancer is refractory or resistant to anti -PD- 1 therapy ⁇ i.e., the cancer does not respond to the therapy). In some embodiments, a cancer is refractory or resistant to anti-PD-Ll therapy (i.e., the cancer does not respond to the therapy).
  • a subject has a cancer that is becoming refractory or resistant to a specified treatment (such as an anti -PD 1 or anti-PD-Ll therapy), e.g., the subject has become less responsive to the treatment since first receiving it.
  • a specified treatment such as an anti -PD 1 or anti-PD-Ll therapy
  • the subject has not received the treatment, but has a type of cancer that does not typically respond to the treatment.
  • A“superficial” lesion or metastasis is a lesion or metastasis that is within the skin or is at the surface of skin. In some embodiments, a superficial lesion or metastasis is within the cutis. In some embodiments, a superficial lesion or metastasis is within the dermis. In some embodiments, a superficial lesion or metastasis is within the epidermis.
  • A“subcutaneous” lesion or metastasis is under the skin.
  • a subcutaneous lesion or metastasis is with the subcutis.
  • a“tumor lesion” or “lesion” is a solid tumor, e.g. , a primary solid tumor or a solid tumor that has arisen from a metastasis from another solid tumor.
  • squamous cell refers to any thin flat cells found, for example, in the surface of the skin, eyes, various internal organs, and the lining of hollow organs and ducts of some glands.
  • CSCC cutaneous squamous cell carcinoma
  • squamous cell carcinoma of the head and neck refers to all stages and all forms of cancer of the head and neck that begin in squamous cells.
  • Squamous cell carcinoma of the head and neck includes (but is not limited to) cancers of the nasal cavity, sinuses, lips, mouth, salivary glands, throat, and larynx (voice box).
  • melanoma refers to all stages and all forms of cancer that begins in melanocytes. Melanoma typically begins in a mole (skin melanoma), but can also begin in other pigmented tissues, such as in the eye or in the intestines.
  • A“tumor-involved regional lymph node” or“tumor-involved node” refers to metastasis- containing regional lymph node.
  • a tumor-involved regional lymph node is a clinically occult tumor-involved regional lymph node.
  • a tumor- involved regional lymph node is a clinically detectable tumor-involved regional lymph node.
  • a “clinically occult” tumor-involved regional lymph node describes microscopically identified regional node metastasis without clinical or radiographic evidence of regional node metastasis.
  • a clinically occult tumor-involved regional lymph node is detected by sentinel lymph node (SLN) biopsy and without clinical or radiographic evidence of regional node metastasis.
  • SSN sentinel lymph node
  • “clinically detectable” nodal metastasis describes patients with regional node metastasis identifiable by clinical, radiographic, or ultrasound examination and usually (but not necessarily) confirmed by biopsy.
  • Non-nodal locoregional sites refer to metastases that are a consequence of
  • intralymphatic or angiotrophic tumor spread include microsatellite, satellite, and in-transit metastases.“Satellite” metastases refer to clinically evident cutaneous and/or subcutaneous metastases occurring within 2 cm of a primary melanoma.
  • “Microsatellite” metastases refer to microscopic cutaneous and/or subcutaneous metastases found adjacent or deep to a primary melanoma on pathological examination of the primary site. In some embodiments, microsatellite metastases are completely discontinuous from a primary melanoma with unaffected stroma occupying the space between.
  • In-transit metastases refer to clinically evident cutaneous and/or subcutaneous metastases identified at a distance more than 2 cm from a primary melanoma in the region between the primary and the first echelon of regional lymph nodes.
  • satellite or in-transmit metastases may occur distal to a primary melanoma.
  • matted nodes refer to two or more nodes adherent to one another through involvement by metastatic disease. In some embodiments, matted nodes are identified at the time a specimen is examined macroscopically in a pathology laboratory.
  • A“distant metastasis” refers to cancer that has spread from the primary tumor to a distant organ or a distant lymph node.
  • the distant metastasis is detectable in skin, subcutaneous tissue, muscle, or distant lymph nodes.
  • the distant metastasis is detectable in a lung.
  • the distant metastasis is detectable in central nerve system (CNS).
  • the distant metastasis is detectable in any other visceral site other than CNS, including the lungs, the heart, or an organ of the digestive, excretory, reproductive, or circulatory system.
  • a distant metastasis is in a tissue or organ that is not in direct contact (e.g ., touching or directly connected to) the tissue or organ containing the primary tumor.
  • a metastasis e.g., a distant metastasis
  • is in e.g, is detectable in the liver.
  • ENE Extranodal extension
  • Cystic metastasis that stretches, but does not breach, the lymph node capsule may be classified as ENE-negative.
  • the ENE-positive includes large extranodal vessels.
  • the ENE-positive extends less than 2 mm from the node capsule. In some embodiments, the ENE-positive extends more than 2 mm from the lymph node capsule or is apparent to the naked eye at dissection.
  • “Deep invasion” refers to as thickness greater than 6 mm or invasion deeper than subcutaneous fat. In some embodiments, invasion is present in nerves greater than 0.1 mm, deeper than the dermis.
  • “Inhibit,”“inhibitory,” and the like refer to a complete or partial block of an interaction, or a reduction in a biological effect.
  • an anti -PD 1 antibody that inhibits binding of PD-1 to PD-L1 may completely or partially block the interaction.
  • Inhibiting suppression of T cell activation includes any amount of a reduction in suppression.
  • Inhibiting tumor growth or metastasis includes reduction or complete cessation.
  • an effective amount refers to an amount of an agent (such as a mixture of RNAs) that provides a desired biological, therapeutic, and/or prophylactic result. That result can be reduction, amelioration, palliation, lessening, delaying, prevention, and/or alleviation of one or more of the signs, symptoms, or causes of a disease (such as advanced stage solid tumor cancer).
  • an effective amount comprises an amount sufficient to cause a solid tumor/lesion to shrink.
  • an effective amount is an amount sufficient to decrease the growth rate of a solid tumor (such as to suppress tumor growth).
  • an effective amount is an amount sufficient to delay tumor development.
  • an effective amount is an amount sufficient to prevent or delay tumor recurrence. In some embodiments, an effective amount is an amount sufficient to increase a subject’s immune response to a tumor, such that tumor growth and/or size and/or metastasis is reduced, delayed, ameliorated, and/or prevented. An effective amount can be administered in one or more administrations.
  • administration of an effective amount may: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, retard, slow to some extent and may stop cancer cell infiltration into peripheral organs; (iv) inhibit (e.g., slow to some extent and/or block or prevent) metastasis; (v) inhibit tumor growth; (vi) prevent or delay occurrence and/or recurrence of tumor; and/or (vii) relieve to some extent one or more of the symptoms associated with the cancer.
  • co-administered or“co-administration” or the like as used herein refers to administration of two or more agents concurrently, simultaneously, or essentially at the same time, either as part of a single formulation or as multiple formulations that are
  • “Essentially at the same time” as used herein means within about 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, or 6 hours period of each other.
  • the RNA comprises a modified nucleobase in place of at least one (e.g, every) uridine. In some embodiments, the RNA comprises a Capl structure at the 5’ end of the RNA. In some embodiments, the RNA comprises a modified nucleobase in place of at least one (e.g, every) uridine and a Capl structure at the 5’ end of the RNA. In some embodiments, the 5’ UTR comprises SEQ ID NOs: 4 or 6. In some embodiments, the RNA has been processed to reduce double-stranded RNA (dsRNA), such as, for example, by purification on cellulose (as described in the Examples and as known in the art), or via high performance liquid chromatography (HPLC). The“Capl” structure may be generated after in- vitro transcription by enzymatic capping or during in-vitro transcription (co-transcriptional capping).
  • dsRNA double-stranded RNA
  • HPLC high performance liquid chromatography
  • the building block cap for modified RNA is as follows, which is used when co-transcriptionally capping:
  • m2 7 ’ 3 °Gppp(mi 2 °)ApG also sometimes referred to as m2 7 ’ 3 °G(5’)ppp(5’)m 2 °ApG
  • m2 7 ’ 3 °Gppp(mi 2 °)ApG also sometimes referred to as m2 7 ’ 3 °G(5’)ppp(5’)m 2 °ApG
  • Capl RNA after co-transcriptional capping which comprises RNA and m2 7 ’ 3 0 G(5’)ppp(5’)m 2 °ApG:
  • the RNA is modified with“CapO” structures generated during in-vitro transcription (co-transcriptional capping) using, in one embodiment, the cap analog anti-reverse cap (ARCA Cap (m2 7 ’ 3 °G(5’)ppp(5’)G)) with the structure:
  • CapO RNA comprising RNA and m2 7 ’ 3 °G(5’)ppp(5’)G:
  • the“CapO” structures are generated during in-vitro transcription (co-transcriptional capping) using the cap analog Beta-S-ARCA
  • uracil describes one of the nucleobases that can occur in the nucleic acid of RNA.
  • the structure of uracil is:
  • uridine describes one of the nucleosides that can occur in RNA.
  • the structure of uridine is:
  • Pseudo-UTP (pseudouridine 5’ -triphosphate) has the following structure:
  • Pseudouridine is one example of a modified nucleoside that is an isomer of uridine, where the uracil is attached to the pentose ring via a carbon-carbon bond instead of a nitrogen-carbon glycosidic bond. Pseudouridine is described, for example, in Charette and Gray, Life ; 49:341-351 (2000).
  • N1 -methyl-pseudouridine (ihIY) which has the structure:
  • m5U 5-methyl-uridine
  • poly-A tail or“poly-A sequence” refers to an uninterrupted or interrupted sequence of adenylate residues which is typically located at the 3' end of an RNA molecule.
  • Poly-A tails or poly-A sequences are known to those of skill in the art and may follow the 3’ UTR in the RNAs described herein.
  • An uninterrupted poly-A tail is characterized by consecutive adenylate residues. In nature, an uninterrupted poly-A tail is typical.
  • RNAs disclosed herein can have a poly-A tail attached to the free 3' end of the RNA by a template-independent RNA polymerase after transcription or a poly-A tail encoded by DNA and transcribed by a template-dependent RNA polymerase.
  • a poly-A tail of about 120 A nucleotides has a beneficial influence on the levels of RNA in transfected eukaryotic cells, as well as on the levels of protein that is translated from an open reading frame that is present upstream (5’) of the poly- A tail (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017).
  • the poly-A tail may be of any length.
  • a poly-A tail comprises, essentially consists of, or consists of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides, and, in particular, about 120 A nucleotides.
  • “essentially consists of’ means that most nucleotides in the poly-A tail, typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% by number of nucleotides in the poly-A tail are A nucleotides, but permits that remaining nucleotides are nucleotides other than A nucleotides, such as U nucleotides (uridylate), G nucleotides (guanylate), or C nucleotides (cytidylate).
  • a poly-A tail is attached during RNA transcription, e.g, during preparation of in vitro transcribed RNA, based on a DNA template comprising repeated dT nucleotides (deoxythymidylate) in the strand complementary to the coding strand.
  • the DNA sequence encoding a poly-A tail (coding strand) is referred to as poly(A) cassette.
  • the poly(A) cassette present in the coding strand of DNA essentially consists of dA nucleotides, but is interrupted by a random sequence of the four nucleotides (dA, dC, dG, and dT). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length.
  • a cassette is disclosed in WO 2016/005324 Al, hereby incorporated by reference. Any poly(A) cassette disclosed in WO 2016/005324 Al may be used in the present invention.
  • a poly(A) cassette that essentially consists of dA nucleotides, but is interrupted by a random sequence having an equal distribution of the four nucleotides (dA, dC, dG, dT) and having a length of e.g. 5 to 50 nucleotides shows, on DNA level, constant propagation of plasmid DNA in E. coli and is still associated, on RNA level, with the beneficial properties with respect to supporting RNA stability and translational efficiency is encompassed. Consequently, in some embodiments, the poly-A tail contained in an RNA molecule described herein essentially consists of A nucleotides, but is interrupted by a random sequence of the four nucleotides (A, C, G, U). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length.
  • nucleotides other than A nucleotides flank a poly-A tail at its 3' end, i.e., the poly-A tail is not masked or followed at its 3' end by a nucleotide other than A.
  • a poly-A tail comprises the sequence:
  • RNA and“mRNA” are used interchangeably, except where the context makes clear that one or the other is appropriate, such as where“mRNA” is appropriate to use to distinguish from other types of RNA (rRNA or tRNA) and where“RNA” is appropriate to refer to the structure of the transcription product prior to the 5’ capping to form a mRNA.
  • IFNa is used generically herein to describe any interferon alpha Type I cytokine, including IFNa2b and IFNa4.
  • treatment covers any administration or application of a therapeutic for disease in a subject, and includes inhibiting the disease, arresting its development, relieving one or more symptoms of the disease, curing the disease, or preventing reoccurrence of the disease.
  • treatment of a solid tumor may comprise alleviating symptoms of the solid tumor, decreasing the size of the solid tumor, eliminating the solid tumor, reducing further growth of the tumor, or reducing or eliminating recurrence of a solid tumor after treatment.
  • Treatment may also be measured as a change in a biomarker of effectiveness or in an imaging or radiographic measure.
  • the term“monotherapy,” as used herein, means a therapy that uses one type of treatment, such as, e.g ., RNA therapy alone, radiation therapy alone, or surgery alone, to treat a certain disease or condition (such as cancer).
  • monotherapy refers to the use of a single drug (which may include multiple active agents, such as, e.g. , a mixture of RNAs) to treat a disease or condition.
  • the monotherapy is a therapy that is administered to treat cancer, without any other therapy that is used to treat the cancer.
  • a monotherapy for treating a cancer may optionally be combined with another treatment to ameliorate a symptom of the cancer but not treat the cancer per se (e.g, the treatment is not intended or expected to impact the growth or size of a solid tumor), but may not be combined with any other therapy directed against the cancer, such as, e.g, a chemotherapeutic agent or radiation therapy.
  • administering a mixture of RNAs as a monotherapy means administering the mixture of RNAs without, e.g, radiation therapy or any
  • administering a mixture of RNAs as a monotherapy does not preclude administering concurrently or simultaneously with the mixture of RNAs, agents that are not directed against the cancer, such as, e.g, agents that reduce pain.
  • prevention means inhibiting or arresting
  • cancer including solid tumors
  • Methodastasis means the process by which cancer spreads from the place at which it first arose as a primary tumor to other locations in the body.
  • the term“intratum orally,” or“intratumoral” as used herein, means into the tumor.
  • intra-tumoral injection means injecting the therapeutic at any location that touches the tumor.
  • Lymphoma is a solid tumor cancer derived from lymphocytes. Lymphoma includes Hodgkin and Non-Hodgkin lymphoma. Lymphoma forms solid
  • tumors/neoplasms within lymph nodes can also be found in non-lymph node tissues when metastasized.
  • peripherally is an area that is about 2-mm wide and is adjacent to the invasive front of the tumor periphery.
  • the peri-tumoral area comprises host tissue. See, for example, Fig. 11.
  • administering means providing a pharmaceutical agent or composition to a subject, and includes, but is not limited to, administering by a medical professional and self- administering.
  • the disclosure describes nucleic acid sequences and amino acid sequences having a certain degree of identity to a given nucleic acid sequence or amino acid sequence, respectively (a reference sequence).
  • sequence identity between two nucleic acid sequences indicates the percentage of nucleotides that are identical between the sequences.
  • Sequence identity between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences.
  • % identical “% identity” or similar terms are intended to refer, in particular, to the percentage of nucleotides or amino acids which are identical in an optimal alignment between the sequences to be compared. Said percentage is purely statistical, and the differences between the two sequences may be but are not necessarily randomly distributed over the entire length of the sequences to be compared. Comparisons of two sequences are usually carried out by comparing the sequences, after optimal alignment, with respect to a segment or “window of comparison”, in order to identify local regions of corresponding sequences. The optimal alignment for a comparison may be carried out manually or with the aid of the local homology algorithm by Smith and Waterman, 1981, Ads App. Math.
  • NCBI National Center for Biotechnology Information
  • the algorithm parameters used for BLASTN algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 28; (iii) Max matches in a query range set to 0; (iv) Match/Mismatch Scores set to 1, -2; (v) Gap Costs set to Linear; and (vi) the filter for low complexity regions being used.
  • the algorithm parameters used for BLASTP algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 3; (iii) Max matches in a query range set to 0; (iv) Matrix set to BLOSUM62; (v) Gap Costs set to Existence: 11 Extension: 1; and (vi) conditional compositional score matrix adjustment.
  • Percentage identity is obtained by determining the number of identical positions at which the sequences to be compared correspond, dividing this number by the number of positions compared (e.g, the number of positions in the reference sequence) and multiplying this result by 100.
  • the degree of identity is given for a region which is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference sequence.
  • the degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides, in some embodiments in continuous nucleotides.
  • the degree of identity is given for the entire length of the reference sequence.
  • Nucleic acid sequences or amino acid sequences having a particular degree of identity to a given nucleic acid sequence or amino acid sequence, respectively, may have at least one functional property of said given sequence, e.g, and in some instances, are functionally equivalent to said given sequence.
  • One important property includes the ability to act as a cytokine, in particular when administered to a subject.
  • a nucleic acid sequence or amino acid sequence having a particular degree of identity to a given nucleic acid sequence or amino acid sequence is functionally equivalent to the given sequence.
  • antibody encompasses various antibody structures, including monoclonal antibodies, polyclonal antibodies, multispecific antibodies (for example, bispecific and trispecific antibodies), and antibody fragments so long as they exhibit the desired activity.
  • antibody includes, fragments that are capable of binding to an antigen, such as Fv, single-chain Fv (scFv), Fab, Fab’, di-scFv, sdAb (single domain antibody) and (Fab’)2 (including a chemically linked F(ab’)2).
  • the term antibody also includes chimeric antibodies and humanized antibodies as long as they are suitable for human administration.
  • Antibody fragments also include either orientation of single chain scFvs, tandem di-scFv, diabodies, tandem tri-sdcFv, minibodies, etc.
  • Antibody fragments also include nanobodies (sdAb, an antibody having a single, monomeric domain, such as a pair of variable domains of heavy chains, without a light chain).
  • the term“monoclonal antibody” refers to an antibody of a substantially homogeneous population of antibodies, that is, the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Thus, a sample of monoclonal antibodies can bind to the same epitope on the antigen.
  • the modifier“monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies may be made by the hybridoma method first described by Kohler and Milstein, 1975, Nature 256:495, or may be made by recombinant DNA methods such as described in U.S. Pat. No. 4,816,567.
  • the monoclonal antibodies may also be isolated from phage libraries generated using the techniques described in McCafferty et al.,
  • CDR denotes a complementarity determining region as defined by at least one manner of identification to one of skill in the art.
  • the various CDRs within an antibody can be designated by their appropriate number and chain type, including, without limitation as: a) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3; b) CDRLl, CDRL2, CDRL3, CDRH1, CDRH2, and CDRH3; c) LCDR-1, LCDR-2, LCDR-3, HCDR-1, HCDR-2, and HCDR-3; or d) LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3; etc.
  • the term“CDR” is used herein to also encompass HVR or a“hyper variable region”, including hypervariable loops ( e.g ., Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987).)
  • the term“heavy chain variable region” as used herein refers to a region comprising at least three heavy chain CDRs.
  • the heavy chain variable region includes the three CDRs and at least FR2 and FR3.
  • the heavy chain variable region includes at least heavy chain HCDR1, framework (FR) 2, HCDR2, FR3, and HCDR3.
  • a heavy chain variable region also comprises at least a portion of an FR1 and/or at least a portion of an FR4.
  • the term“heavy chain constant region” as used herein refers to a region comprising at least three heavy chain constant domains, CHI, CH2, and CH3.
  • Nonlimiting exemplary heavy chain constant regions include g, d, and a.
  • Nonlimiting exemplary heavy chain constant regions also include e and m.
  • Each heavy constant region corresponds to an antibody isotype.
  • an antibody comprising a g constant region is an IgG antibody
  • an antibody comprising a d constant region is an IgD antibody
  • an antibody comprising an a constant region is an IgA antibody.
  • an antibody comprising a m constant region is an IgM antibody
  • an antibody comprising an e constant region is an IgE antibody.
  • IgG antibodies include, but are not limited to, IgGl (comprising a yl constant region), IgG2 (comprising a g2 constant region), IgG3 (comprising a g3 constant region), and IgG4 (comprising a g4 constant region) antibodies
  • IgA antibodies include, but are not limited to, IgAl (comprising an al constant region) and IgA2 (comprising an a.2 constant region) antibodies
  • IgM antibodies include, but are not limited to, IgMl and IgM2.
  • the term“heavy chain” as used herein refers to a polypeptide comprising at least a heavy chain variable region, with or without a leader sequence.
  • a heavy chain comprises at least a portion of a heavy chain constant region.
  • full-length heavy chain refers to a polypeptide comprising a heavy chain variable region and a heavy chain constant region, with or without a leader sequence.
  • the term“light chain variable region” as used herein refers to a region comprising at least three light chain CDRs.
  • the light chain variable region includes the three CDRs and at least FR2 and FR3.
  • the light chain variable region includes at least light chain LCDR1, framework (FR) 2, LCDR2, FR3, and LCDR3.
  • a light chain variable region may comprise light chain CDR1, framework (FR) 2, CDR2, FR3, and CDR3.
  • a light chain variable region also comprises at least a portion of an FR1 and/or at least a portion of an FR4.
  • light chain constant region refers to a region comprising a light chain constant domain, CL.
  • Nonlimiting exemplary light chain constant regions include X and .
  • non-function-altering deletions and alterations within the domains are encompassed within the scope of the term“light chain constant region,” unless designated otherwise.
  • the term“light chain” as used herein refers to a polypeptide comprising at least a light chain variable region, with or without a leader sequence. In some embodiments, a light chain comprises at least a portion of a light chain constant region.
  • the term“full-length light chain” as used herein refers to a polypeptide comprising a light chain variable region and a light chain constant region, with or without a leader sequence.
  • epitope refers to a site on a target molecule to which an antibody binds. Epitopes often include a chemically active surface grouping of molecules such as amino acids, polypeptides or sugar side chains and have specific three-dimensional structural characteristics as well as specific charge characteristics.
  • transitional term“comprising”, which is synonymous with“including,”“containing,” or“characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.
  • transitional phrase“consisting of’ excludes any element, step, or component not specified in the claim, and the transitional phrase“consisting essentially of’ limits the scope of the claim term to the recited components and those that do not materially affect the basic and novel characteristics of the claimed term, as understood from the specification.
  • methods for treating advanced-stage solid tumor cancers comprising administering to a subject having an advanced- stage solid tumor cancer RNA encoding an IL-12sc protein, RNA encoding an IL-15 sushi protein, RNA encoding an IFNa protein, and RNA encoding a GM-CSF protein in combination with an anti- PD-1 antibody. Details of the administered RNA follow.
  • administering RNAs comprises administering RNA encoding IFNa, RNA encoding IL-15 sushi, RNA encoding IL-12sc, and RNA encoding GM- CSF, optionally modified to have a modified nucleobase in place of each uridine and a Capl structure at the 5’ end of the RNA.
  • administering RNAs comprises administering RNA encoding IL-12sc and further administering an RNA encoding IFNa, IL-15 sushi, and GM-CSF.
  • administering RNAs comprises administering RNA encoding IFNa and further administering an RNA encoding IL-12sc, IL-15 sushi, and GM-CSF.
  • administering RNAs comprises administering RNA encoding IL-15 sushi and further administering an RNA encoding IL-12sc, IFNa, and GM-CSF.
  • administering RNAs comprises administering RNA encoding GM-CSF sushi and further administering an RNA encoding IL-12sc, IFNa, and IL-15 sushi.
  • the IFNa protein in the cytokine RNA mixture is an IFNa2b protein
  • the method comprises administering RNA encoding an IFNa2b protein.
  • the RNA encoding an IL-12sc protein comprises the nucleotide sequence of SEQ ID NO: 17 or 18, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:
  • the IL-12sc protein comprises the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 14.
  • the RNA encoding an IL-15 sushi protein comprises the nucleotide sequence of SEQ ID NO: 26, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 26 and/or (ii) the IL-15 sushi protein comprises the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 24.
  • the RNA encoding an IFNa protein comprises the nucleotide sequence of SEQ ID NO: 22 or 23, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 22 or 23 and/or (ii) the IFNa protein comprises the amino acid sequence of SEQ ID NO: 19, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 19.
  • the RNA encoding a GM-CSF protein comprises the nucleotide sequence of SEQ ID NO: 29, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 29 and/or (ii) the GM-CSF protein comprises the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 27.
  • IL-12sc Interleukin-12 single-chain
  • an RNA that encodes interleukin- 12 single-chain is provided.
  • the interleukin- 12 single-chain (IL-12sc) RNA is encoded by a DNA sequence encoding interleukin- 12 single-chain (IL-12sc) (e.g ., SEQ ID NO: 14), which comprises IL-12 p40 (sometimes referred to as IL-12B; encoded by nucleotides 1- 984 of SEQ ID NO: 15), a linker, such as a GS linker, and IL-12 p35 (sometimes referred to as IL-12A; encoded by nucleotides 1027-1623 of SEQ ID NO: 15).
  • IL-12sc interleukin- 12 single-chain
  • the IL- 12p40, linker, and IL-12p35 are consecutive with no intervening nucleotides.
  • An exemplary DNA sequence encoding IL-12sc is provided in SEQ ID NO: 15.
  • the interleukin- 12 single-chain (IL-12sc) RNA is provided at SEQ ID NO: 17 or 18, both of which encode the protein of SEQ ID NO: 14.
  • the RNA sequence of IL-12 p40 is shown at nucleotides 1-984 of SEQ ID NO: 17 or 18 and the RNA sequence of IL-12 p35 is shown at nucleotides 1027-1623 of SEQ ID NO: 17 or 18.
  • the IL-12sc RNA is encoded by a codon-optimized DNA sequence encoding IL-12sc. In some embodiments, the IL-12sc RNA is encoded by a codon- optimized DNA sequence encoding IL-12 p40. In some embodiments, the IL-12sc RNA is encoded by a codon-optimized DNA sequence encoding IL-12 p35. In some embodiments, the codon-optimized DNA sequence comprises or consists of SEQ ID NO: 16. In some
  • the DNA sequence comprises a codon-optimized DNA sequence with 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 16.
  • the codon-optimized DNA sequence encoding IL-12 p40 comprises the nucleotides encoding the IL-12sc-p40 (nucleotides 1-984 of SEQ ID NO: 16).
  • the codon-optimized DNA sequence encoding IL-12 p35 comprises the nucleotides encoding the IL-12sc-p35 (nucleotides 1027-1623 of SEQ ID NO: 16).
  • the codon-optimized DNA sequence encoding IL-12sc comprises the nucleotides encoding the IL-12sc-p40 (nucleotides 1-984 of SEQ ID NO: 16) and -p35 (nucleotides 1027- 1623 of SEQ ID NO: 16) portions of SEQ ID NO: 16 and further comprises nucleotides between the p40 and p35 portions ( e.g ., nucleotides 985-1026 of SEQ ID NO: 16) encoding a linker polypeptide connecting the p40 and p35 portions. Any linker known to those of skill in the art may be used.
  • the p40 portion may be 5’ or 3’ to the p35 portion.
  • the IL-12sc RNA comprises an RNA sequence that is, for example, transcribed from a DNA sequence encoding IL-12sc.
  • the RNA may also be recombinantly produced.
  • the RNA sequence is transcribed from a nucleotide sequence comprising SEQ ID NOs: 15 or 16.
  • the RNA sequence comprises or consists of SEQ ID NOs: 17 or 18.
  • the RNA sequence comprises or consists of an RNA sequence with 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NOs: 17 or 18.
  • the RNA sequence comprises the nucleotides encoding the IL-12sc-p40 (nucleotides 1-984 of SEQ ID NOs: 17 or 18) and -p35 (nucleotides 1027-1623 of SEQ ID NOs: 17 or 18) portions of SEQ ID NOs: 17 or 18.
  • the codon-optimized RNA sequence encoding IL- 12sc comprises the nucleotides encoding the IL-12sc-p40 (nucleotides 1-984 of SEQ ID NO:
  • one or more uridine in the IL-12sc RNA is replaced by a modified nucleoside as described herein.
  • the modified nucleoside replacing uridine is pseudouridine (y), N1 -methyl-pseudouridine (m ) or 5 -methyl -uridine (m 5 U).
  • the RNA comprises a modified nucleoside in place of each uridine.
  • the modified nucleoside is N1 -methyl-pseudouridine (mV).
  • the IL-12sc RNA comprises an altered nucleotide at the 5’ end.
  • the RNA comprises a 5’ cap. Any 5’ cap known in the art may be used.
  • the 5’ cap comprises a 5’ to 5’ triphosphate linkage.
  • the 5’ cap comprises a 5’ to 5’ triphosphate linkage including thiophosphate modification.
  • the 5’ cap comprises a 2 -O or 3'-0-ribose-methylated nucleotide.
  • the 5’ cap comprises a modified guanosine nucleotide or modified adenosine nucleotide.
  • the 5’ cap comprises 7-methylguanylate. In some embodiments, the 5’ cap is CapO or Capl.
  • Exemplary cap structures include m7G(5’)ppp(5’)G, m7,2O-mG(5’)ppsp(5’)G, m7G(5 , )ppp(5 , )2'0-mG, and m7,3'0- mG(5’)ppp(5’ )2' O- m A.
  • the IL-12sc RNA comprises a 5’ untranslated region (UTR).
  • the 5’ UTR is upstream of the initiation codon.
  • the 5’ UTR regulates translation of the RNA.
  • the 5’ UTR is a stabilizing sequence.
  • the 5’ UTR increases the half-life of RNA. Any 5’ UTR known in the art may be used.
  • the 5’ UTR RNA sequence is transcribed from SEQ ID NOs: 3 or 5.
  • the 5’ UTR RNA sequence comprises or consists of SEQ ID NOs: 4 or 6.
  • the 5’ UTR RNA sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 4 or 6.
  • the IL-12sc RNA comprises a 3’ UTR.
  • the 3’ UTR follows the translation termination codon.
  • the 3’ UTR regulates polyadenylation, translation efficiency, localization, or stability of the RNA.
  • the 3’ UTR RNA sequence is transcribed from SEQ ID NO: 7. In some embodiments, the 3’ UTR RNA sequence comprises or consists of SEQ ID NO: 8. In some embodiments, the 3’ UTR RNA sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8.
  • the IL-12sc RNA comprises both a 5’ UTR and a 3’ UTR. In some embodiments, the IL-12sc RNA comprises only a 5’ UTR. In some embodiments, the IL-12sc RNA comprises only a 3’ UTR.
  • the IL-12sc RNA comprises a poly-A tail.
  • the RNA comprises a poly-A tail of at least about 25, at least about 30, at least about 50 nucleotides, at least about 70 nucleotides, or at least about 100 nucleotides.
  • the poly-A tail comprises 200 or more nucleotides.
  • the poly-A tail comprises or consists of SEQ ID NO: 30.
  • the RNA comprises a 5’ cap, a 5’ UTR, a nucleic acid encoding IL-12sc, a 3’ UTR, and a poly-A tail, in that order.
  • the IL-12sc RNA is encoded by a DNA sequence comprising or consisting of a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 15 or 16 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 3 or 5.
  • the IL-12sc RNA comprises an RNA sequence that is, for example, transcribed from a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 15 or 16 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 3 or 5.
  • the RNA may also be recombinantly produced.
  • one or more uridine in the IL-12sc RNA is replaced by a modified nucleoside as described herein.
  • the modified nucleoside replacing uridine is pseudouridine (y), N1 -methyl-pseudouridine 5-methyl-uridine (m 5 U).
  • the RNA comprises a modified nucleoside in place of each uridine.
  • the modified nucleoside is N1 -methyl-pseudouridine (m ) ⁇
  • the IL-12sc RNA is encoded by a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 15 or 16 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.
  • the IL-12sc RNA comprises an RNA sequence that is, for example, transcribed from a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 15 or 16 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.
  • the RNA may also be recombinantly produced.
  • one or more uridine in the IL-12sc RNA is replaced by a modified nucleoside as described herein.
  • the modified nucleoside replacing uridine is pseudouridine (y), N1 -methyl-pseudouridine (m ) or 5-methyl-uridine (m 5 U).
  • the RNA comprises a modified nucleoside in place of each uridine.
  • the modified nucleoside is N1 -methyl-pseudouridine (m ).
  • the IL-12sc RNA is encoded by a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 15 or 16; at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 3 or 5; and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.
  • the IL-12sc RNA comprises an RNA sequence that is, for example, transcribed from a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 15 or 16; at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 3 or 5; and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,
  • the RNA may also be recombinantly produced.
  • one or more uridine in the IL-12sc RNA is replaced by a modified nucleoside as described herein.
  • the modified nucleoside replacing uridine is pseudouridine (y), N1 -methyl-pseudouridine (m ) or 5 -methyl -uridine (m 5 U).
  • the RNA comprises a modified nucleoside in place of each uridine.
  • the modified nucleoside is N1 -methyl-pseudouridine (mV) ⁇
  • the IL-12sc RNA comprises an RNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 17 or 18; at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 4 or 6; and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8.
  • one or more uridine in the IL-12sc RNA is replaced by a modified nucleoside as described herein.
  • the modified nucleoside replacing uridine is pseudouridine (y), N1 -methyl-pseudouridine (m ) or 5-methyl-uridine (m 5 U).
  • the interferon alpha (IFNa) RNA is encoded by a DNA sequence encoding interferon alpha (IFNa) ( e.g ., SEQ ID NO: 19).
  • An exemplary DNA sequence encoding this IFNa is provided in SEQ ID NO: 20.
  • the IFNa RNA is encoded by a codon-optimized DNA sequence encoding IFNa.
  • the codon-optimized DNA sequence comprises or consists of the nucleotides of SEQ ID NO: 21.
  • the DNA sequence comprises or consists of a codon-optimized DNA sequence with 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 21.
  • the IFNa RNA comprises an RNA sequence that is, for example, transcribed from a DNA sequence encoding IFNa.
  • the RNA may also be
  • the RNA sequence is transcribed from a nucleotide sequence comprising SEQ ID NOs: 20 or 21.
  • the RNA sequence comprises or consists of SEQ ID NOs: 22 or 23.
  • the RNA sequence comprises or consists of an RNA sequence with 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NOs: 22 or 23.
  • one or more uridine in the IFNa RNA is replaced by a modified nucleoside as described herein.
  • the modified nucleoside replacing uridine is pseudouridine (y), N1 -methyl-pseudouridine (m ) or 5 -methyl -uridine (m 5 U).
  • each uridine in the RNA is modified.
  • each uridine in the RNA is modified with N1 -methyl-pseudouridine (mV) ⁇
  • the IFNa RNA comprises an altered nucleotide at the 5’ end.
  • the IFNa RNA comprises a 5’ cap. Any 5’ cap known in the art may be used.
  • the 5’ cap comprises a 5’ to 5’ triphosphate linkage.
  • the 5’ cap comprises a 5’ to 5’ triphosphate linkage including
  • the 5’ cap comprises a 2 -O or 3'-0-ribose- methylated nucleotide. In some embodiments, the 5’ cap comprises a modified guanosine nucleotide or modified adenosine nucleotide. In some embodiments, the 5’ cap comprises 7- methylguanylate. In some embodiments, the 5’ cap is CapO or Capl.
  • Exemplary cap structures include m7G(5’)ppp(5’)G, m7,2O-mG(5’)ppsp(5’)G, m7G(5’)ppp(5’)2O-mG and m7,3O- mG(5’)ppp(5’ )2' O- m A.
  • the IFNa RNA comprises a 5’ untranslated region (UTR).
  • the 5’ UTR is upstream of the initiation codon.
  • the 5’ UTR regulates translation of the RNA.
  • the 5’ UTR is a stabilizing sequence.
  • the 5’ UTR increases the half-life of RNA. Any 5’ UTR known in the art may be used.
  • the 5’ UTR RNA sequence is transcribed from a nucleotide sequence comprising SEQ ID NOs: 3 or 5.
  • the 5’ UTR RNA sequence comprises or consists of SEQ ID NOs: 4 or 6.
  • UTR RNA sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 4 or 6.
  • the IFNa RNA comprises a 3’ UTR.
  • the 3’ UTR follows the translation termination codon. In some embodiments, the 3’ UTR regulates polyadenylation, translation efficiency, localization, or stability of the RNA.
  • the 3’ UTR RNA sequence is transcribed from a nucleotide sequence comprising SEQ ID NO: 7. In some embodiments, the 3’ UTR RNA sequence comprises or consists of SEQ ID NO: 8. In some embodiments, the 3’ UTR RNA sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8.
  • the IFNa RNA comprises both a 5’ UTR and a 3’ UTR.
  • the composition comprises only a 5’ UTR. In some embodiments, the composition comprises only a 3’ UTR.
  • the IFNa RNA comprises a poly-A tail.
  • the IFNa RNA comprises a poly-A tail of at least about 25, at least about 30, at least about 50 nucleotides, at least about 70 nucleotides, or at least about 100 nucleotides.
  • the poly-A tail comprises 200 or more nucleotides.
  • the poly-A tail comprises or consists of SEQ ID NO: 30.
  • the RNA comprises a 5’ cap, a 5’ UTR, a nucleic acid encoding IFNa, a 3’ UTR, and a poly-A tail, in that order.
  • the IFNa RNA is encoded by a DNA sequence comprising or consisting of a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 20 or 21 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 3 or 5.
  • the IFNa RNA comprises an RNA sequence that is, for example, transcribed from a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 20 or 21 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 3 or 5.
  • the RNA may also be recombinantly produced.
  • one or more uridine in the IFNa RNA is replaced by a modified nucleoside as described herein.
  • the modified nucleoside replacing uridine is pseudouridine (y), N1 -methyl-pseudouridine (m ) or 5-methyl-uridine (m 5 U).
  • the RNA comprises a modified nucleoside in place of each uridine.
  • the modified nucleoside is N1 -methyl-pseudouridine (m ).
  • the IFNa RNA is encoded by a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 20 or 21 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.
  • the IFNa RNA comprises an RNA sequence that is, for example, transcribed from a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 20 or 21 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.
  • one or more uridine in the IFNa RNA is replaced by a modified nucleoside as described herein.
  • the modified nucleoside replacing uridine is pseudouridine (y), N1 -methyl-pseudouridine (m ) or 5-methyl- uridine (m 5 U).
  • the RNA comprises a modified nucleoside in place of each uridine.
  • the modified nucleoside is N1 -methyl-pseudouridine (mV).
  • the IFNa RNA is encoded by a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 20 or 21; at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 3 or 5; and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.
  • the IFNa RNA comprises an RNA sequence that is, for example, transcribed from a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 20 or 21; at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 3 or 5; and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.
  • the RNA may also be recombinantly produced.
  • one or more uridine in the IFNa RNA is replaced by a modified nucleoside as described herein.
  • the modified nucleoside replacing uridine is pseudouridine (y), N1 -methyl-pseudouridine (mV) or 5 -methyl -uridine (m 5 U).
  • the RNA comprises a modified nucleoside in place of each uridine.
  • the modified nucleoside is N1 -methyl-pseudouridine (mV) -
  • the composition comprises an RNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 22 or 23; at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 4 or 6; and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8.
  • one or more uridine in the IFNa RNA is replaced by a modified nucleoside as described herein.
  • the modified nucleoside replacing uridine is pseudouridine (y), N1 -methyl- pseudouridine (mV) or 5 -methyl-uridine (m 5 U).
  • an RNA that encodes an interleukin- 15 (IL-15) sushi is administered.
  • the term“IL-15 sushi” describes a construct comprising the soluble interleukin 15 (IL-15) receptor alpha sushi domain and mature interleukin alpha (IL-15) as a fusion protein.
  • the IL-15 sushi RNA is encoded by a DNA sequence encoding IL-15 sushi (SEQ ID NO: 24), which comprises the soluble IL-15 receptor alpha chain (sushi) followed by a glycine-serine (GS) linker followed by the mature sequence of IL-15.
  • SEQ ID NO: 24 DNA sequence encoding IL-15 sushi
  • GS glycine-serine
  • the IL-15 sushi RNA is an RNA sequence that is, for example, transcribed from a DNA sequence encoding IL-15 sushi.
  • the RNA may also be recombinantly produced.
  • the RNA sequence is transcribed from a nucleotide sequence comprising SEQ ID NO: 25.
  • the nucleotides encoding the linker may be completely absent or replaced in part or in whole with any nucleotides encoding a suitable linker.
  • the RNA sequence comprises or consists of SEQ ID NO: 26.
  • the RNA sequence comprises an RNA sequence with 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 26.
  • the DNA or RNA sequence encoding IL-15 sushi comprises the nucleotides encoding the sushi domain of IL-15 receptor alpha (e.g ., nucleotide 1-321 of SEQ ID NOs: 25 or 26) and mature IL-15 (e.g., nucleotide 382-729 of SEQ ID NO: 25 or 26).
  • the DNA or RNA sequence encoding IL-15 sushi comprises the nucleotides encoding the sushi domain of IL-15 receptor alpha (e.g., nucleotide 1-321 of SEQ ID NOs: 25 or 26) and mature IL-15 (e.g, nucleotide 382-729 of SEQ ID NOs: 25 or 26) and further comprises nucleotides between these portions encoding a linker polypeptide connecting the portions.
  • the linker comprises nucleotides 322-381 of SEQ ID Nos: 25 or 26. Any linker known to those of skill in the art may be used.
  • one or more uridine in the IL-15 sushi RNA is replaced by a modified nucleoside as described herein.
  • the modified nucleoside replacing uridine is pseudouridine (y), N1 -methyl-pseudouridine (m ) or 5 -methyl -uridine (m 5 U).
  • the RNA comprises a modified nucleoside in place of each uridine.
  • the modified nucleoside is N1 -methyl-pseudouridine (mV).
  • the IL-15 sushi RNA comprises an altered nucleotide at the 5’ end.
  • the IL-15 sushi RNA comprises a 5’ cap. Any 5’ cap known in the art may be used.
  • the 5’ cap comprises a 5’ to 5’ triphosphate linkage.
  • the 5’ cap comprises a 5’ to 5’ triphosphate linkage including thiophosphate modification.
  • the 5’ cap comprises a 2 -O or 3'-0-ribose- methylated nucleotide.
  • the 5’ cap comprises a modified guanosine nucleotide or modified adenosine nucleotide.
  • the 5’ cap comprises 7- methylguanylate. In some embodiments, the 5’ cap is CapO or Capl.
  • Exemplary cap structures include m7G(5’)ppp(5’)G, m7,2O-mG(5’)ppsp(5’)G, m7G(5’)ppp(5’)2O-mG and m7,3O- mG(5’)ppp(5’ )2' O- m A.
  • the IL-15 sushi RNA comprises a 5’ untranslated region (UTR).
  • the 5’ UTR is upstream of the initiation codon.
  • the 5’ UTR regulates translation of the RNA.
  • the 5’ UTR is a stabilizing sequence.
  • the 5’ UTR increases the half-life of RNA. Any 5’ UTR known in the art may be used.
  • the 5’ UTR RNA sequence is transcribed from SEQ ID NOs: 3 or 5.
  • the 5’ UTR RNA sequence comprises or consists of SEQ ID NOs: 4 or 6.
  • the 5’ UTR RNA sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 4 or 6.
  • the IL-15 sushi RNA comprises a 3’ UTR.
  • the 3’ UTR follows the translation termination codon.
  • the 3’ UTR regulates polyadenylation, translation efficiency, localization, or stability of the RNA.
  • the 3’ UTR RNA sequence is transcribed from SEQ ID NO: 7. In some embodiments, the 3’ UTR RNA sequence comprises or consists of SEQ ID NO: 8. In some embodiments, the 3’ UTR RNA sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8.
  • the IL-15 sushi RNA comprises both a 5’ UTR and a 3’ UTR. In some embodiments, the IL-15 sushi RNA comprises only a 5’ UTR. In some embodiments, the IL-15 sushi RNA comprises only a 3’ UTR.
  • the IL-15 sushi RNA comprises a poly-A tail.
  • the RNA comprises a poly-A tail of at least about 25, at least about 30, at least about 50 nucleotides, at least about 70 nucleotides, or at least about 100 nucleotides.
  • the poly-A tail comprises 200 or more nucleotides.
  • the poly-A tail comprises or consists of SEQ ID NO: 30.
  • the RNA comprises a 5’ cap, a 5’ UTR, a nucleic acid encoding IL-15 sushi, a 3’ UTR, and a poly- A tail, in that order.
  • the IL-15 sushi RNA is encoded by a DNA sequence comprising or consisting of a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 25 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 3 or 5.
  • the IL-15 sushi RNA comprises an RNA sequence that is, for example, transcribed from a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 25 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 3 or 5.
  • the RNA may also be recombinantly produced.
  • one or more uridine in the IFNa RNA is replaced by a modified nucleoside as described herein.
  • the modified nucleoside replacing uridine is pseudouridine (y), N1 -methyl-pseudouridine (m ) or 5-methyl-uridine (m 5 U).
  • the RNA comprises a modified nucleoside in place of each uridine.
  • the modified nucleoside is N1 -methyl-pseudouridine (m ).
  • the IL-15 sushi RNA comprises a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 25 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.
  • the IL-15 sushi RNA comprises an RNA sequence that is, for example, transcribed from a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 25 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.
  • the RNA may also be recombinantly produced.
  • one or more uridine in the IFNa RNA is replaced by a modified nucleoside as described herein.
  • the modified nucleoside replacing uridine is pseudouridine (y), N1 -methyl-pseudouridine (m ) or 5-methyl-uridine (m 5 U).
  • the RNA comprises a modified nucleoside in place of each uridine.
  • the modified nucleoside is N1 -methyl-pseudouridine (mV) ⁇
  • the IL-15 sushi RNA comprises a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 25; at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 3 or 5; and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.
  • the IL-15 sushi RNA comprises an RNA sequence that is, for example, transcribed from a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 25; at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 3 or 5; and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,
  • one or more uridine in the IFNa RNA is replaced by a modified nucleoside as described herein.
  • the modified nucleoside replacing uridine is pseudouridine (y), N1 -methyl-pseudouridine (m ) or 5-methyl-uridine (m 5 U).
  • the RNA comprises a modified nucleoside in place of each uridine.
  • the modified nucleoside is N1 -methyl- pseudouridine (mV).
  • the IL-15 sushi RNA comprises an RNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 26; at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 4 or 6; and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8.
  • one or more uridine in the IFNa RNA is replaced by a modified nucleoside as described herein.
  • the modified nucleoside replacing uridine is pseudouridine (y), N1 -methyl-pseudouridine (m ) or 5-methyl-uridine (m 5 U).
  • GM-CSF Granulocyte-macrophage colony-stimulating factor
  • an RNA that encodes granulocyte-macrophage colony- stimulating factor is administered.
  • the GM-CSF RNA is encoded by a DNA sequence encoding granulocyte-macrophage colony-stimulating factor (GM- CSF) (e.g ., SEQ ID NO: 27).
  • the DNA sequence encoding GM-CSF is provided in SEQ ID NO: 28.
  • the GM-CSF RNA comprises an RNA sequence that is, for example, transcribed from a DNA sequence encoding GM-CSF.
  • the RNA sequence is transcribed from SEQ ID NO: 28.
  • the RNA may also be recombinantly produced.
  • the RNA sequence comprises or consists of SEQ ID NO: 29.
  • the RNA sequence comprises an RNA sequence with 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NOs: 29.
  • one or more uridine in the GM-CSF RNA is replaced by a modified nucleoside as described herein.
  • the modified nucleoside replacing uridine is pseudouridine (y), N1 -methyl-pseudouridine (m ) or 5 -methyl -uridine (m 5 U).
  • the RNA comprises a modified nucleoside in place of each uridine.
  • the modified nucleoside is N1 -methyl-pseudouridine (m ) ⁇
  • the GM-CSF RNA comprises an altered nucleotide at the 5’ end.
  • the RNA comprises a 5’ cap.
  • the 5’ cap comprises a 5’ to 5’ triphosphate linkage. In some embodiments, the 5’ cap comprises a 5’ to 5’ triphosphate linkage including thiophosphate modification. In some embodiments, the 5’ cap comprises a 2 -O or 3' -O-ribose-m ethylated nucleotide. In some embodiments, the 5’ cap comprises a modified guanosine nucleotide or modified adenosine nucleotide. In some embodiments, the 5’ cap comprises 7-methylguanylate. In some embodiments,
  • the 5’ cap is CapO or Capl.
  • Exemplary cap structures include m7G(5’)ppp(5’)G, m7,2' 0-mG(5’ )ppsp(5’ )G, m7G(5’ )ppp(5’ )2' O-mG and m7,3 ' O- mG(5’ )ppp(5’ )2' O-m A.
  • the GM-CSF RNA comprises a 5’ untranslated region (UTR).
  • the 5’ UTR is upstream of the initiation codon.
  • the 5’ UTR regulates translation of the RNA.
  • the 5’ UTR is a stabilizing sequence.
  • the 5’ UTR increases the half-life of RNA. Any 5’ UTR known in the art may be used.
  • the 5’ UTR RNA sequence is transcribed from SEQ ID NOs: 3 or 5.
  • the 5’ UTR RNA sequence comprises or consists of SEQ ID NOs: 4 or 6.
  • the 5’ UTR RNA sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 4 or 6.
  • the GM-CSF RNA comprises a 3’ UTR.
  • the 3’ UTR follows the translation termination codon.
  • the 3’ UTR regulates polyadenylation, translation efficiency, localization, or stability of the RNA.
  • the 3’ UTR RNA sequence is transcribed from SEQ ID NO: 7. In some embodiments, the 3’ UTR RNA sequence comprises or consists of SEQ ID NO: 8. In some embodiments, the 3’ UTR RNA sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8.
  • the GM-CSF RNA comprises both a 5’ UTR and a 3’ UTR. In some embodiments, the RNA comprises only a 5’ UTR. In some embodiments, the composition comprises only a 3’ UTR.
  • the GM-CSF RNA comprises a poly-A tail.
  • the RNA comprises a poly-A tail of at least about 25, at least about 30, at least about 50 nucleotides, at least about 70 nucleotides, or at least about 100 nucleotides.
  • the poly-A tail comprises 200 or more nucleotides.
  • the poly-A tail comprises or consists of SEQ ID NO: 30.
  • the GM-CSF RNA comprises a 5’ cap, a 5’ UTR, nucleotides encoding GM-CSF, a 3’ UTR, and a poly-A tail, in that order.
  • the GM-CSF RNA is encoded by a DNA sequence comprising or consisting of a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 28 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 3 or 5.
  • the GM-CSF RNA comprises an RNA sequence that is, for example, transcribed from a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 28 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 3 or 5.
  • the RNA may also be recombinantly produced.
  • one or more uridine in the GM-CSF RNA is replaced by a modified nucleoside as described herein.
  • the modified nucleoside replacing uridine is pseudouridine (y), N1 -methyl-pseudouridine (m ) or 5-methyl-uridine (m 5 U).
  • the RNA comprises a modified nucleoside in place of each uridine.
  • the modified nucleoside is N1 -methyl-pseudouridine (m ).
  • the GM-CSF RNA is encoded by a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 28 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.
  • the GM-CSF RNA comprises an RNA sequence that is, for example, transcribed from a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 28 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.
  • the RNA may also be recombinantly produced.
  • one or more uridine in the GM-CSF RNA is replaced by a modified nucleoside as described herein.
  • the modified nucleoside replacing uridine is pseudouridine (y), N1 -methyl-pseudouridine (m ) or 5-methyl-uridine (m 5 U).
  • the RNA comprises a modified nucleoside in place of each uridine.
  • the modified nucleoside is N1 -methyl-pseudouridine (m ).
  • the GM-CSF RNA comprises a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 28; at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 3 or 5; and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.
  • the GM-CSF RNA comprises an RNA sequence that is, for example, transcribed from a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 28; at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 3 or 5; and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,
  • the RNA may also be recombinantly produced.
  • one or more uridine in the GM-CSF RNA is replaced by a modified nucleoside as described herein.
  • the modified nucleoside replacing uridine is pseudouridine (y), N1 -methyl-pseudouridine (m ) or 5 -methyl -uridine (m 5 U).
  • the RNA comprises a modified nucleoside in place of each uridine.
  • the modified nucleoside is N1 -methyl-pseudouridine (mV).
  • the GM-CSF RNA comprises an RNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 29; at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 4 or 6; and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8.
  • one or more uridine in the GM-CSF RNA is replaced by a modified nucleoside as described herein.
  • the modified nucleoside replacing uridine is pseudouridine (y), N1 -methyl-pseudouridine (m ) or 5-methyl-uridine (m 5 U).
  • each of the RNAs described herein may be modified in any way known to those of skill in the art.
  • each RNA is modified as follows:
  • the 5’ UTR comprises SEQ ID NOs: 4 or 6.
  • the RNA has been processed to reduce double-stranded RNA (dsRNA) as described above.
  • dsRNA double-stranded RNA
  • The“Capl” structure may be generated after in-vitro transcription by enzymatic capping or during in-vitro transcription (co-transcriptional capping).
  • one or more uridine in the RNA is replaced by a modified nucleoside.
  • the modified nucleoside is a modified uridine.
  • the modified uridine replacing uridine is pseudouridine ( y), N1 -methyl-pseudouridine (m 1 y), or 5-methyl-uridine (m5U).
  • one or more cytosine, adenine or guanine in the RNA is replaced by modified nucleobase(s).
  • the modified nucleobase replacing cytosine is 5-methylcytosine (m 5 C).
  • the modified nucleobase replacing adenine is N 6 -methyladenine (m 6 A).
  • any other modified nucleobase known in the art for reducing the immunogenicity of the molecule can be used.
  • the modified nucleoside replacing one or more uridine in the RNA may be any one or more of 3-methyl-uridine (m 3 U), 5-methoxy-uridine (mo 5 U), 5- aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s 2 U), 4-thio-uridine (s 4 U), 4- thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho 5 U), 5-aminoallyl-uridine, 5- halo-uridine ( e.g ., 5-iodo-uridineor 5-bromo-uridine), uridine 5-oxyacetic acid (cmo 5 U), uridine 5-oxyacetic acid methyl ester (mcmo 5 U), 5-carboxymethyl -uridine (cm 5 U), 1-carboxymethyl- pseudouridine, 5-carboxyhydroxymethyl-uridine (chm 5 U), 5-
  • At least one RNA comprises a modified nucleoside in place of at least one uridine. In some embodiments, at least one RNA comprises a modified nucleoside in place of each uridine. In some embodiments, each RNA comprises a modified nucleoside in place of at least one uridine. In some embodiments, each RNA comprises a modified nucleoside in place of each uridine.
  • the modified nucleoside is independently selected from pseudouridine (y), N1 -methyl-pseudouridine (m 1 y), and 5-methyl-uridine (m5U).
  • the modified nucleoside comprises pseudouridine (y).
  • the modified nucleoside comprises N1 -methyl-pseudouridine (ih ⁇ y).
  • the modified nucleoside comprises 5-methyl-uridine (m5U).
  • At least one RNA may comprise more than one type of modified nucleoside, and the modified nucleosides are independently selected from pseudouridine (y), N1 -methyl-pseudouridine (m 1 y), and 5- methyl-uridine (m5U).
  • the modified nucleosides comprise pseudouridine (y) and N1 -methyl-pseudouridine (ih ⁇ y).
  • the modified nucleosides comprise pseudouridine (y) and 5-methyl-uridine (m5U).
  • the modified nucleosides comprise N1 -methyl -pseudouridine (ih ⁇ y) and 5-methyl-uridine (m5U).
  • the modified nucleosides comprise pseudouridine (y), N1 -methyl-pseudouridine (ih ⁇ y), and 5-methyl-uridine (m5U).
  • At least one RNA used in the method comprises the 5’ cap m2 7 ’ 3 °Gppp(mi 2 °)ApG or 3'-0-Me-m 7 G(5')ppp(5')G.
  • each RNA used in the method comprises the 5’ cap m2 7 ’ 3 °Gppp(mi 2 °)ApG or 3'-0-Me-m 7 G(5')ppp(5')G.
  • each RNA used in the method comprises the 5’ cap m2 7 ’ 3 °Gppp(mi 2 °)ApG.
  • each RNA used in the method comprises the 3 -O-Me- m 7 G(5')ppp(5')G. In some embodiments, each RNA used in the method comprises the 5’ cap m2 7 ’ 3 °Gppp(mi 2 °)ApG and 3'-0-Me-m 7 G(5')ppp(5')G.
  • At least one RNA comprises a 5’ UTR comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 4 and 6, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 4 and 6.
  • each RNA comprises a 5’ UTR comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 4 and 6, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 4 and 6.
  • At least one RNA comprises a 3’ UTR comprising the nucleotide sequence of SEQ ID NO: 8, or a nucleotide sequence having at least 99%, 98%,
  • each RNA comprises a 3’ UTR comprising the nucleotide sequence of SEQ ID NO: 8, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 8.
  • at least one RNA comprises a poly-A tail.
  • each RNA comprises a poly-A tail.
  • the poly-A tail may comprise at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly-A tail may essentially consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides. In some embodiments, the poly-A tail may consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides.
  • the poly-A tail may comprise the poly-A tail shown in SEQ ID NO: 30. In some embodiments, the poly-A tail comprises at least 100 nucleotides. In some embodiments, the poly-A tail comprises about 150 nucleotides. In some embodiments, the poly-A tail comprises about 120 nucleotides.
  • one or more RNA comprises: (1) a 5’ cap comprising m2 7 ’ 3 °Gppp(mi 2 °)ApG or 3'-0-Me-m 7 G(5')ppp(5')G; (2) a 5’ UTR comprising (i) a nucleotide sequence selected from the group consisting of SEQ ID NOs: 4 and 6, or (ii) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 4 and 6; (3) a 3’ UTR comprising (i) the nucleotide sequence of SEQ ID NO: 8, or (ii) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:8; and
  • Cancer cells engage multiple mechanisms to evade anti-tumor host immune responses, including expression of programmed cell death- 1 ligand 1 (PD-L1), the primary ligand for programmed cell death 1 receptor (PD-1), which is expressed on activated B and T lymphocytes and myeloid cells. Interaction of PD-L1 with PD-1 results in decreased immune responses and contributes to tumor evasion.
  • An anti-PD-1 antibody is an antibody that binds to PD-1 and inhibits the interaction of PD-1 with PD-L1. Upon administration to a subject, an anti- PD-1 antibody may bind to PD-1, inhibit its binding to PD-L1, and prevent the activation of its downstream signaling pathways, including activation of T cells.
  • the cytokine RNA mixture is administered in combination with an anti -PD 1 antibody.
  • an anti -PD 1 antibody Encompassed herein are anti -PD 1 antibodies that inhibit the interaction of PD-1 with PD-L1, and inhibit the suppression of an immune response that is triggered when PD-1 interacts with PD-L1.
  • whether or not an anti -PD 1 antibody inhibits the suppression of an immune response is assessed by measuring T-cell activation (sometimes also referred to as T-cell proliferation). Such measurement may be assessed in vivo (e.g, after administration of an anti -PD 1 antibody to a human subject) or in vitro (e.g, in a cell-based assay). In some embodiments, the ability of an anti -PD 1 antibody to inhibit the suppression of an immune response is determined in a cell-based assay using either engineered T cell lines or primary human T cells according to the methods of Burova et al. (2017) Mol. Cancer 16(5); 861-70.
  • human PD-1 protein and a reporter are expressed in T cells and the T cells are activated with, e.g, with an anti-CD3 x anti-CD20 bispecific antibody.
  • Antigen-presenting cells such as HEK293 cells, are generated to express human CD20 and human PD-L1.
  • Serially diluted test anti -PD 1 antibody is applied and the expression of the reporter is analyzed.
  • the anti -PD 1 antibody inhibits the suppression of an immune response that is triggered when PD-1 interacts with PD-L1 by at least 70%, at least 80%, at least 90%, or at least 95% as compared to the inhibition seen with cemiplimab.
  • whether an antibody inhibits the suppression of an immune response that is triggered when PD-1 interacts with PD-L1 by at least 70%, at least 80%, at least 90%, or at least 95% as compared to the inhibition seen with cemiplimab is assessed by measuring T-cell activation as described herein.
  • the anti -PD 1 antibody is a chimeric, humanized or human antibody. In some embodiments, the anti-PD-1 antibody is isolated and/or recombinant. In some embodiments, the anti -PD 1 antibody is a multi-specific antibody such as, for example, a tri- specific or bi-specific antibody.
  • Non-limiting examples of anti-PD-1 antibodies include cemiplimab (see, e.g., U.S. Pat. No. 9,987,500 B2, also referred to as REGN2810, see e.g. CAS Number 1801342-60- 8, and Falchook et al. J Immunother Cancer. 2016 Nov;4:70), nivolumab (see, e.g., U.S. Pat.
  • pembrolizumab see, e.g., U.S. Pat. No. 8,354,509
  • MEDI0608 formerly AMP-514; see, e.g., U.S. Pat. No. 8,609,089 and U.S. Patent No.
  • spartalizumab also known as PDR001, (see, e.g., WO 2015/112900), PF-06801591 (see, e.g., WO 2016/092419), and tislelizumab (also known as BGB-A317, (see, e.g., WO 2015/035606), camrelizumab (also known as SHR-1210; see e.g., WO 2015/085847), dostarlimab (also known as TSR-042; see, e.g., WO 2014/179664), sintilimab (also known as IBI308; see, e.g., WO 2017/025016), JS001 (see, e.g., WO 2014/206107), MGA012 (see, e.g., WO 2017/019846), AGEN2034 (see, e.g., WO 2017/040790), and JNJ-63723283
  • Cemiplimab includes Cemiplimab-rwlc.
  • the anti-PD-1 antibody is one of those disclosed in WO 2015/112800 (such as those referred to as H1M7789N, H1M7799N, H1M7800N, H2M7780N, H2M7788N, H2M7790N, H2M7791N, H2M7794N, H2M7795N, H2M7796N, H2M7798N, H4H9019P, H4xH9034P2, H4xH9035P2, H4xH9037P2, H4xH9045P2, H4xH9048P2,
  • H4xH9145P2, H4xH8992P, H4xH8999P and H4xH9008P in Table 1 of the PCT publication and those referred to as H4H7798N, H4H7795N2, H4H9008P and H4H9048P2 in Table 3 of the PCT publication).
  • the disclosure of WO 2015/112800 is incorporated by reference herein in its entirety.
  • the antibodies disclosed in WO 2015/112800 and related antibodies including antibodies and antigen-binding fragments having the CDRs, VH and VL sequences, or heavy and light chain sequences disclosed in that PCT publication, as well as antibodies and antigen-binding fragments binding to the same PD-1 epitope as the antibodies disclosed in that PCT publication, can be used in conjunction with the RNA cytokine mixture to treat and/or prevent cancer.
  • the anti-PD-1 antibody comprises Pidilizumab (also referred to as CT-011) (Berger et ah, 2008. Clin Cancer Res. 14(10):3044-51), PF-06801591 (ClinicalTrials.gov identifier: NCT02573259), mDX-400 (Merck & Co), MEDI0680 (also referred to as AMP-514) (ClinicalTrials.gov Identifier: NCT02013804), PDR001
  • the anti -PD-1 antibody comprises the heavy and light chain amino acid sequences shown below as SEQ ID NOs: 31 and 32, respectively; the VH and
  • VL sequences in SEQ ID Nos: 39 and 40 (shown in italics), or one or more (e.g., all six) CDRs in SEQ ID Nos: 31 and 32 (shown in bold boxes).
  • an antibody comprising the following CDRs is encompassed:
  • HCDR1 GFTFSNFG (SEQ ID NO: 33)
  • HCDR2 ISGGGRDT (SEQ ID NO: 34)
  • HCDR3 VKWGNIYFDY (SEQ ID NO: 35)
  • LCDR1 LSINTF (SEQ ID NO: 36)
  • LCDR2 AAS (SEQ ID NO: 37)
  • LCDR3 QQSSNTPFT (SEQ ID NO: 38).
  • the anti-PD-1 antibody comprises HCDR3 (SEQ ID NO: 35). In some embodiments, the anti-PD-1 antibody comprises LCDR3 (SEQ ID NO: 38). In some embodiments, the anti-PD-1 antibody comprises HCDR3 (SEQ ID NO: 35) and LCDR3 (SEQ ID NO: 38).
  • the anti-PDl antibody comprises HCDR3 (SEQ ID NO: 35) and/or LCDR3 (SEQ ID NO: 38), and inhibits the interaction of PD-1 with PD-L1.
  • the anti-PDl antibody comprises HCDR3 (SEQ ID NO: 35) and/or LCDR3 (SEQ ID NO: 38), and inhibits the suppression of an immune response that is triggered when PD-1 interacts with PD-L1.
  • the anti-PDl antibody comprises HCDR3 (SEQ ID NO: 35) and/or LCDR3 (SEQ ID NO: 38), and inhibits the interaction of PD-1 with PD-L1, and inhibits the suppression of an immune response that is triggered when PD-1 interacts with PD-L1.
  • HCDRl GFTFSNFG (SEQ ID NO: 33)
  • HCDR2 ISGGGRDT (SEQ ID NO: 34)
  • HCDR3 VKWGNIYFDY (SEQ ID NO: 35)
  • LCDR2 AAS (SEQ ID NO: 37)
  • the anti-PDl antibody is cemiplimab. In some embodiments, the anti-PDl antibody is an antibody that binds to the same epitope as cemiplimab. In some embodiments, the anti-PDl antibody competes with cemiplimab for PD-1 binding. In some embodiments, whether an antibody competes with cemiplimab for PD-1 binding is determined via ELISA according to methods known to those of skill in the art, e.g ., as in Burova et al. (2017) Mol. Cancer 16(5); 861-70.
  • a test antibody, cemiplimab, and a negative isotype control antibody are incubated with PD-1 and transferred to the wells of an ELISA plate coated with PD-L1. Bound antibody is detected after appropriate washing and application of labelled secondary antibody.
  • the anti-PDl antibody inhibits the interaction of PD-1 with PD-L1 by at least 70%, at least 80%, at least 90%, or at least 95% as compared to the level of inhibition seen with cemiplimab. In some embodiments, whether an antibody inhibits the interaction of PD-1 with PD-L1 by at least 70%, at least 80%, at least 90%, or at least 95% as compared to the level of inhibition seen with cemiplimab is assessed via ELISA as described herein.
  • Cemiplimab is currently being investigated in phase 1 clinical studies as monotherapy and in combination with other anti-cancer therapies, as well as in phase 2 and 3 clinical studies in patients with advanced cutaneous squamous cell carcinoma, basal cell carcinoma, non-small cell lung cancer, cervical cancer, and other solid tumors. Preliminary efficacy was observed in several tumor types, including non-small cell lung cancer, at both 1 mg/kg administered every 2 weeks (Q2W) and 3 mg/kg Q2W doses.
  • cemiplimab As of 27 March 2018, 757 patients were enrolled and treated with cemiplimab as monotherapy as well as in combination with radiation therapy and/or other cancer therapy at different dose levels (1, 3, or 10 mg/kg or 200 mg Q2W; and 3 mg/kg, 250 mg, or 350 mg Q3W).
  • the efficacy of cemiplimab against advanced CSCC has been clearly documented in a phase 2 study, and on 28 September 2018 cemiplimab received approval in the United States for the treatment of patients with metastatic CSCC or locally advanced CSCC who are not candidates for curative surgery or curative radiation. Cemiplimab was also approved in Europe for the same indication on June 28, 2019.
  • Cemiplimab has a safety profile similar to that of other PD-1 inhibitors.
  • TEAEs treatment-emergent adverse events
  • compositions and methods of using and making Cemiplimab are disclosed, for example, in the published U.S. Patent Application No. 2015/0203579, the content of which is hereby incorporated by reference herein in its entirety for any purpose.
  • the anti -PD- 1 antibody may be formulated with suitable carriers, excipients, and other agents that provide suitable transfer, delivery, tolerance, and the like.
  • suitable carriers excipients, and other agents that provide suitable transfer, delivery, tolerance, and the like.
  • formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles, DNA conjugates, anhydrous absorption pastes, oil-in water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See Powell et al.“Compendium of excipients for parenteral formulations” PDA (1998) J Pharm Sci Technol 52:238-311.
  • the cytokine RNA mixture provided herein may be used in methods, e.g ., therapeutic methods, in combination with an anti -PD- 1 antibody.
  • methods for treating advanced-stage, unresectable, or metastatic solid tumor cancers comprising administering the cytokine RNA mixture and an anti-PD-1 antibody, wherein the advanced-stage solid tumor cancer comprises an epithelial tumor, prostate tumor, ovarian tumor, renal cell tumor, gastrointestinal tract tumor, hepatic tumor, colorectal tumor, tumor with vasculature, mesothelioma tumor, pancreatic tumor, breast tumor, sarcoma tumor, lung tumor, colon tumor, melanoma tumor, small cell lung tumor, neuroblastoma tumor, testicular tumor, carcinoma tumor, adenocarcinoma tumor, seminoma tumor, retinoblastoma, cutaneous squamous cell carcinoma (CSCC), lymphoma, including Non-Hodgkin lymph
  • the advanced- stage solid tumor cancer comprises an epithelial tumor, prostate tumor, ovarian tumor, renal cell tumor, gastrointestinal tract tumor, hepatic tumor, colorectal tumor, tumor with vasculature, mesothelioma tumor, pancreatic tumor, breast tumor, sarcoma tumor, lung tumor, colon tumor, melanoma tumor, small cell lung tumor, neuroblastoma tumor, testicular tumor, carcinoma tumor, adenocarcinoma tumor, seminoma tumor, retinoblastoma, cutaneous squamous cell carcinoma (CSCC), squamous cell carcinoma for the head and neck (HNSCC), head and neck cancer, osteosarcoma tumor, non-small cell lung cancer, kidney tumor, thyroid tumor, liver tumor, other solid tumors amenable to intratumoral injection, or combinations thereof.
  • CSCC cutaneous squamous cell carcinoma
  • HNSCC head and neck cancer
  • osteosarcoma tumor non-small cell lung cancer, kidney tumor, thyroid tumor, liver
  • the advanced-stage solid tumor cancer comprises lymphoma, such as Non-Hodgkin lymphoma or Hodgkin lymphoma.
  • the solid tumor cancer is melanoma. In some embodiments, the solid tumor cancer is melanoma. In some
  • the melanoma is uveal melanoma or mucosal melanoma.
  • the solid tumor cancer is melanoma, optionally uveal melanoma or mucosal melanoma, and comprises superficial, subcutaneous and/or lymph node metastases amenable for intratumoral injection.
  • intratumoral injection comprises injection into a solid tumor metastasis within a lymph node. In some embodiments, intratumoral injection comprises injection into a lymphoma tumor within a lymph node. In some embodiments, intratumoral injection comprises injection into a primary or secondary solid tumor that is within 10 cm of the subject’s skin surface. In some embodiments, intratumoral injection comprises injection into a primary or secondary solid tumor that is within 5 cm of the subject’s skin surface. In some embodiments, intratumoral injection comprises injection into a cutaneous solid tumor. In some embodiments, the cutaneous solid tumor is a metastasis. In some embodiments, the cutaneous solid tumor is a skin cancer. In some embodiments, the cutaneous solid tumor is not a skin cancer.
  • intratumoral injection comprises injection into a subcutaneous solid tumor.
  • the subcutaneous solid tumor is a metastasis.
  • the subcutaneous solid tumor is a skin cancer. In some embodiments, the subcutaneous solid tumor is not a skin cancer.
  • the solid tumor is an epithelial tumor. In some embodiments, the solid tumor is an epithelial tumor.
  • the solid tumor is a prostate tumor. In some embodiments, the solid tumor is an ovarian tumor. In some embodiments, the solid tumor is a renal cell tumor. In some
  • the solid tumor is a gastrointestinal tract tumor. In some embodiments, the solid tumor is a hepatic tumor. In some embodiments, the solid tumor is a colorectal tumor. In some embodiments, the solid tumor is a tumor with vasculature. In some embodiments, the solid tumor is a mesothelioma tumor. In some embodiments, the solid tumor is a pancreatic tumor. In some embodiments, the solid tumor is a breast tumor. In some embodiments, the solid tumor is a sarcoma tumor. In some embodiments, the solid tumor is a lung tumor. In some embodiments, the solid tumor is a colon tumor. In some embodiments, the solid tumor is a melanoma tumor.
  • the solid tumor is a small cell lung tumor. In some embodiments, the solid tumor is non-small cell lung cancer tumor. In some embodiments, the solid tumor is a neuroblastoma tumor. In some embodiments, the solid tumor is a testicular tumor. In some embodiments, the solid tumor is a carcinoma tumor. In some embodiments, the solid tumor is an adenocarcinoma tumor. In some embodiments, the solid tumor is a seminoma tumor. In some embodiments, the solid tumor is a retinoblastoma. In some embodiments, the solid tumor is a cutaneous squamous cell carcinoma (CSCC). In some embodiments, the solid tumor is a squamous cell carcinoma for the head and neck (HNSCC).
  • CSCC cutaneous squamous cell carcinoma
  • HNSCC head and neck
  • the solid tumor is HNSCC. In some embodiments, the solid tumor is head and neck cancer. In some embodiments, the solid tumor is an osteosarcoma tumor. In some embodiments, the solid tumor is kidney cancer. In some embodiments, the solid tumor is thyroid cancer. In some
  • the solid tumor is anaplastic thyroid cancer (ATC). In some embodiments, the solid tumor is liver cancer. In some embodiments, the solid tumor is a colon tumor. In some embodiments, the solid tumor is any two of the above. In some embodiments, the solid tumor is any two or more of the above.
  • ATC anaplastic thyroid cancer
  • the solid tumor is liver cancer. In some embodiments, the solid tumor is a colon tumor. In some embodiments, the solid tumor is any two of the above. In some embodiments, the solid tumor is any two or more of the above.
  • the solid tumor is lymphoma. In some embodiments, the solid tumor is Non-Hodgkin lymphoma. In some embodiments, the solid tumor is Hodgkin lymphoma.
  • the method comprises the use of a cytokine RNA mixture comprising RNA encoding IFNa, RNA encoding IL-15 sushi, RNA encoding IL-12sc, and RNA encoding GM-CSF, optionally modified to have a modified nucleobase in place of each uridine and a Capl structure at the 5’ end of the RNA, in combination with an anti -PD- 1 antibody.
  • a method for treating an advanced-stage, unresectable, or metastatic solid tumor cancer comprising administering to a subject having an advanced-stage, unresectable, or metastatic solid tumor cancer RNA encoding an IL-12sc protein, RNA encoding an IL-15 sushi protein, RNA encoding an IFNa protein, and RNA encoding a GM-CSF protein, in combination with an anti -PD- 1 antibody.
  • methods for treating advanced-stage, unresectable, or metastatic solid tumor cancers comprising administering to a subject having an advanced-stage solid tumor cancer a therapeutically effective amount of RNA comprising RNA encoding an IL-12sc protein, RNA encoding an IL-15 sushi protein, RNA encoding an IFNa protein, and RNA encoding a GM-CSF protein and a therapeutically effective amount of an anti -PD- 1 antibody.
  • a method for in treating advanced-stage, unresectable, or metastatic solid tumor cancers comprising administering RNA encoding IL- 12sc and further administering an RNA encoding IFNa, IL-15 sushi, and GM-CSF, and further administering an anti -PD- 1 antibody.
  • a method for treating advanced-stage, unresectable, or metastatic solid tumor cancers comprising administering RNA encoding IFNa and further administering an RNA encoding IL-12sc, IL-15 sushi, and GM-CSF, and further administering an anti -PD- 1 antibody.
  • a method for treating advanced-stage, unresectable, or metastatic solid tumor cancers comprising administering RNA encoding IL-15 sushi and further administering an RNA encoding IL-12sc, IFNa, and GM-CSF, and further administering an anti -PD- 1 antibody.
  • a method for treating advanced-stage, unresectable, or metastatic solid tumor cancers comprising administering RNA encoding GM- CSF sushi and further administering an RNA encoding IL-12sc, IFNa, and IL-15 sushi, and further administering an anti -PD- 1 antibody.
  • methods for treating advanced-stage, unresectable, or metastatic solid tumor cancers comprising administering to a subject having an advanced-stage solid tumor cancer a therapeutically effective amount of 1) RNA comprising RNA encoding an IL-12sc protein, RNA encoding an IL-15 sushi protein, RNA encoding an IFNa protein, and RNA encoding a GM-CSF protein and 2) an anti -PD- 1 antibody.
  • “a/an/the RNAs/anti-PD-1 antibody combination” refers to administering RNA cytokine mixture in combination with an anti -PD 1 antibody.
  • the co-administration of the RNAs and the an anti -PD- 1 antibody result in one or more of: (a) a reduction in the severity or duration of a symptom of cancer; (b) inhibition of tumor growth, or an increase in tumor necrosis, tumor shrinkage and/or tumor disappearance; (c) delay in tumor growth and/or development; (d) inhibited or retarded or stopped tumor metastasis; (e) prevention or delay of recurrence of tumor growth; (f) increase in survival of a subject; and/or (g) a reduction in the use or need for conventional anti -cancer therapy (e.g., reduced or eliminated use of chemotherapeutic or cytotoxic agents) as compared to an untreated subject or a subject administered the RNAs or the anti -PD- 1 antibody as monotherapy.
  • conventional anti -cancer therapy e.g., reduced or eliminated use of chemotherapeutic or cytotoxic agents
  • cytokine RNA mixture and anti-PD-1 antibody is administered in combination with one or more other treatment options (e.g., chemotherapeutic agents, including another immune stimulator, immunotherapy, or checkpoint modulator; or radiation).
  • chemotherapeutic agents including another immune stimulator, immunotherapy, or checkpoint modulator; or radiation.
  • the RNAs or the cytokine RNA mixture are delivered via injection into the tumor (e.g., intratumorally), or near the tumor (peri-tumorally) and the anti- PD1 antibody is delivered in the same manner or systemically, for example, intravenous, enteral or parenteral, including, via injection, infusion, and implantation.
  • the RNAs and antibody may be co-administered, e.g., concurrently, simultaneously or sequentially. If sequential,
  • administration can be in any order and at any appropriate time intervals known to those of skill in the art.
  • the RNAs are injected intratumorally or peritum orally and the anti-PD-1 antibody is administered intravenously. In some embodiments, the RNAs are injected intratumorally and the anti-PD-1 antibody is administered intravenously.
  • the cytokine RNA mixture is administered intratumorally once per week in a 3- or 4-week cycle (i.e., three doses every 21 or four doses every 28 days) and the anti -PD 1 antibody is administered systemically, e.g, intravenously only one time during this 21- or 28-day cycle, optionally on the first day of treatment.
  • the cytokine RNA mixture is administered intratum orally or peri turn orally once per week with an anti-PDl antibody administered intravenously on day 1 of a 3-week cycle (i.e., three doses of the cytokine RNA mixture and one dose of an anti-PDl antibody every 21 days).
  • the cytokine RNA mixture is administered intratumorally or peritumorally once per week with an anti-PDl antibody administered intravenously on day 1 of a 4-week cycle (i.e., four doses of the cytokine RNA mixture and one dose of an anti-PDl antibody every 28 days).
  • intratum oral injection continues weekly until the second tumor assessment, at which time a change of the dose interval of the cytokine RNA mixture to every three weeks may be made.
  • the RNAs and the anti -PD- 1 antibody are administered at the same dosing frequency (e.g ., dosed together or separately on the same days).
  • the RNAs and the anti-PD-1 antibody are administered at a different dosing frequency (e.g., on different days). In some embodiments, the RNAs are administered once every week, and the anti-PD-1 antibody is administered once every three weeks.
  • the cytokine RNA mixture and anti-PDl are co administered on a 3- or 4- week cycle, wherein the cytokine RNA mixture is administered once every week, and the anti-PDl antibody is administered only once.
  • the cytokine RNA mixture and anti-PDl are co administered on a 3- or 4- week cycle, wherein the cytokine RNA mixture is administered once every 2 weeks, and the anti-PDl antibody is administered only once. In some embodiments, the cytokine RNA mixture and anti-PDl are co- administered on a 3- or 4- week cycle, wherein the cytokine RNA mixture is administered once every 3 weeks, and the anti-PDl antibody is administered only once.
  • the cytokine RNA mixture and anti-PDl are co administered on a 3- or 4- week cycle, wherein the cytokine RNA mixture is administered once every 4 weeks, and the anti-PDl antibody is administered only once.
  • combinations of RNA are administered as a 1 : 1 : 1 : 1 ratio based on equal RNA mass (i.e., 1 : 1 : 1 : 1 % (w/w/w/w)).
  • the RNAs/anti-PD-1 antibody combination described herein are administered for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. In some embodiments, the RNAs/anti-PD-1 antibody combination is administered for about 8 months. In some embodiments, the RNAs/anti-PD-1 antibody combination is administered for a maximum of 52 weeks.
  • the anti -PD 1 antibody is administered via injection. In some embodiments, the anti -PD 1 antibody is administered intravenously. In some
  • the anti -PD- 1 antibody is administered intravenously once every three weeks and the cytokine RNA mixture is administered intratumorally or peri-tumorally once every week.
  • the anti -PD- 1 antibody is administered once every three weeks intravenously and the cytokine RNA mixture is administered once every week intra- or peri-tumorally.
  • the RNAs are administered in a therapeutically effective amount.
  • the anti -PD- 1 antibody is administered in a therapeutically effective amount.
  • the therapeutically effective amount is an amount that differs from the therapeutically effective amount for each component individually as monotherapy.
  • the anti -PD 1 antibody is administered at a dose from about 0.05 mg to about 600 mg, e.g., about 0.05 mg, about 0.1 mg, about 1.0 mg, about 1.5 mg, about 2.0 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about
  • 200 mg of an anti -PD- 1 antibody is administered.
  • 240 mg of an anti -PD- 1 antibody is administered.
  • 350 mg of an anti-PD-1 antibody is administered.
  • the anti-PD-1 antibody is cemiplimab and 350 mg of cemiplimab is administered.
  • the amount of anti -PD- 1 antibody contained within the individual doses may be expressed in terms of milligrams of antibody per kilogram of subject body weight ( i.e ., mg/kg).
  • anti -PD- 1 antibody used in the methods described herein may be administered to a subject at a dose of about 0.0001 to about 100 mg/kg of subject body weight.
  • anti-PD-1 antibody may be administered at dose of about 0.1 mg/kg to about 20 mg/kg of a patient's body weight.
  • the anti-PD-1 antibody is cemiplimab and is administered at dose of about 3 mg/kg of a patient's body weight.
  • multiple doses of an anti-PD-1 may be administered to a subject over a defined time course.
  • a method comprises sequentially administering to a subject one or more doses of an anti-PD-1 antibody.
  • sequentially administering means that each dose of the antibody is administered to the subject at a different point in time, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks or months).
  • the methods comprise sequentially administering to the patient a single initial dose of an anti-PD-1 antibody, followed by one or more secondary doses of the anti-PD-1 antibody, and optionally followed by one or more tertiary doses of the anti-PD-1 antibody.
  • the terms“initial dose,”“secondary doses,” and“tertiary doses,” refer to the temporal sequence of administration.
  • the“initial dose” is the dose which is administered at the beginning of the treatment regimen (also referred to as the“baseline dose”);
  • the “secondary doses” are the doses which are administered after the initial dose;
  • the“tertiary doses” are the doses which are administered after the secondary doses.
  • the initial, secondary, and tertiary doses may all contain the same amount of the antibody (anti-PD-1 antibody). In certain embodiments, however, the amount contained in the initial, secondary and/or tertiary doses varies from one another (e.g., adjusted up or down as appropriate) during the course of treatment.
  • one or more (e.g., 1, 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as“loading doses” followed by subsequent doses that are administered on a less frequent basis (e.g.,“maintenance doses”).
  • an anti-PD-1 antibody may be administered to a patient at a loading dose of about 1-3 mg/kg followed by one or more maintenance doses of about 0.1 to about 20 mg/kg of the patient's body weight.
  • each secondary and/or tertiary dose is administered 1 ⁇ 2 to
  • the immediately preceding dose means, in a sequence of multiple administrations, the dose of anti-PD-1 antibody which is administered to a patient prior to the administration of the very next dose in the sequence with no intervening doses.
  • the methods may comprise administering to a patient any number of secondary and/or tertiary doses of an anti-PD-1 antibody. For example, in some embodiments, only a single secondary dose is administered to the patient. In other words, only a single secondary dose is administered to the patient.
  • two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the patient.
  • only a single tertiary dose is administered to the patient.
  • two or more (e.g, 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to the patient.
  • each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1 to 2 weeks after the immediately preceding dose. Similarly, in some embodiments involving multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2 to 4 weeks after the immediately preceding dose. Alternatively, the frequency at which the secondary and/or tertiary doses are administered to a patient can vary over the course of the treatment regimen. The frequency of administration may also be adjusted during the course of treatment by a physician depending on the needs of the individual patient following clinical examination.
  • one or more doses of an anti-PD-1 antibody are administered at the beginning of a treatment regimen as“induction doses” on a more frequent basis (twice a week, once a week or once in 2 weeks) followed by subsequent doses
  • consolidation doses or“maintenance doses” that are administered on a less frequent basis (e.g, once in 4-12 weeks).
  • the RNAs are administered in a neoadjuvant setting.
  • “Neoadjuvant setting” refers to a clinical setting in which the method is carried out before the primary/ definitive therapy (e.g., before surgical resection of the tumor).
  • the anti-PD-1 antibody is administered at a dose from about 0.05 mg to about 600 mg, e.g., about 0.05 mg, about 0.1 mg, about 1.0 mg, about 1.5 mg, about 2.0 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about
  • 200 mg of an anti-PD-1 antibody is administered. In some embodiments, 240 mg of an anti-PD-1 antibody is administered. In some embodiments, 350 mg of an anti-PD-1 antibody is administered. In some embodiments, the anti-PD-1 antibody is cemiplimab and 350 mg of cemiplimab is administered.
  • anti-PD-1 antibody contained within the individual doses may be expressed in terms of milligrams of antibody per kilogram of subject body weight (z.e., mg/kg).
  • anti-PD-1 antibody used in the methods described herein may be administered to a subject at a dose of about 0.0001 to about 100 mg/kg of subject body weight.
  • anti-PD-1 antibody may be administered at dose of about 0.1 mg/kg to about 20 mg/kg of a patient's body weight.
  • the anti-PD-1 antibody is cemiplimab and is administered at dose of about 3 mg/kg of a patient's body weight.
  • multiple doses of an anti-PD-1 may be administered to a subject over a defined time course.
  • a method comprises sequentially administering to a subject one or more doses of an anti-PD-1 antibody.
  • sequentially administering means that each dose of the antibody is administered to the subject at a different point in time, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks or months).
  • the methods comprise sequentially administering to the patient a single initial dose of an anti-PD-1 antibody, followed by one or more secondary doses of the anti-PD-1 antibody, and optionally followed by one or more tertiary doses of the anti-PD-1 antibody.
  • the terms“initial dose,”“secondary doses,” and“tertiary doses,” refer to the temporal sequence of administration.
  • the“initial dose” is the dose which is administered at the beginning of the treatment regimen (also referred to as the“baseline dose”);
  • the “secondary doses” are the doses which are administered after the initial dose;
  • the“tertiary doses” are the doses which are administered after the secondary doses.
  • the initial, secondary, and tertiary doses may all contain the same amount of the antibody (anti-PD-1 antibody). In certain embodiments, however, the amount contained in the initial, secondary and/or tertiary doses varies from one another (e.g., adjusted up or down as appropriate) during the course of treatment.
  • one or more (e.g., 1, 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as“loading doses” followed by subsequent doses that are administered on a less frequent basis (e.g.,“maintenance doses”).
  • an anti-PD-1 antibody may be administered to a patient at a loading dose of about 1-3 mg/kg followed by one or more maintenance doses of about 0.1 to about 20 mg/kg of the patient's body weight.
  • each secondary and/or tertiary dose is administered 1 ⁇ 2 to
  • the immediately preceding dose means, in a sequence of multiple administrations, the dose of anti-PD-1 antibody which is administered to a patient prior to the administration of the very next dose in the sequence with no intervening doses.
  • the methods may comprise administering to a patient any number of secondary and/or tertiary doses of an anti-PD-1 antibody. For example, in some embodiments, only a single secondary dose is administered to the patient. In other words, only a single secondary dose is administered to the patient.
  • two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the patient.
  • only a single tertiary dose is administered to the patient.
  • two or more (e.g, 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to the patient.
  • each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1 to 2 weeks after the immediately preceding dose. Similarly, in some embodiments involving multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2 to 4 weeks after the immediately preceding dose. Alternatively, the frequency at which the secondary and/or tertiary doses are administered to a patient can vary over the course of the treatment regimen. The frequency of administration may also be adjusted during the course of treatment by a physician depending on the needs of the individual patient following clinical examination.
  • one or more doses of an anti-PD-1 antibody are administered at the beginning of a treatment regimen as“induction doses” on a more frequent basis (twice a week, once a week or once in 2 weeks) followed by subsequent doses
  • consolidation doses or“maintenance doses” that are administered on a less frequent basis (e.g ., once in 4-12 weeks).
  • the RNAs/anti-PD-1 antibody combination provided herein is used in a method of treating a subject having a solid tumor.
  • the subject :
  • PD-1 programmed cell death 1
  • PD- Ll anti-programmed cell death 1 ligand
  • ii. has a PD-1 and/or PD-L1 resistant solid tumor
  • iii. has acquired resistance to an anti-PD-1 and/or anti-PD-Ll therapy; and/or iv. has innate resistance to anti-PD-1 and/or anti-PD-Ll therapy.
  • the RNAs/anti-PD-1 antibody combination provided herein is used in a method of treating a solid tumor in a subject that has failed an anti programmed cell death 1 (PD-1) or anti-programmed cell death 1 ligand (PD-L1) therapy.
  • PD-1 programmed cell death 1
  • PD-L1 anti-programmed cell death 1 ligand
  • the RNAs/anti-PD-1 antibody combination provided herein is used in a method of treating a solid tumor in a subject that has become intolerant to an anti-programmed cell death 1 (PD-1) or anti-programmed cell death 1 ligand (PD-L1) therapy.
  • PD-1 anti-programmed cell death 1
  • PD-L1 anti-programmed cell death 1 ligand
  • the RNAs/anti-PD-1 antibody combination provided herein is used in a method of treating a solid tumor in a subject that has become intolerant an anti-programmed cell death 1 (PD-1) or anti-programmed cell death 1 ligand (PD-L1) therapy.
  • PD-1 anti-programmed cell death 1
  • PD-L1 anti-programmed cell death 1 ligand
  • the RNAs/anti-PD-1 antibody combination provided herein is used in a method of treating a solid tumor in a subject that has a PD-1 and/or PD-L1 resistant solid tumor.
  • the RNAs/anti-PD-1 antibody combination provided herein is used in a method of treating a solid tumor in a subject, wherein the subject has acquired resistance to an anti-PD-1 and/or anti-PD-Ll therapy.
  • the RNAs/anti-PD-1 antibody combination provided herein is used in a method of treating a solid tumor in a subject, wherein the subject has innate resistance to an anti-PD-1 and/or anti-PD-Ll therapy.
  • the subject has a metastatic solid tumor. In some embodiments, the subject has an unresectable solid tumor. In some embodiments, the subject has an advanced-stage solid tumor. In some embodiments, the subject has a metastatic solid tumor cancer. In some embodiments, the subject has an advanced stage, unresectable, and metastatic solid tumor. In some embodiments, the subject has an advanced stage and unresectable solid tumor. In some embodiments, the subject has an advanced stage and metastatic solid tumor. In some embodiments, the subject has an unresectable and metastatic solid tumor.
  • the subject has a cancer cell comprising a partial or total loss of beta-2-microglobulin (B2M) function.
  • B2M beta-2-microglobulin
  • the subject has a cancer cell with a partial loss of B2M function.
  • the subject has a cancer cell has a total loss of B2M function.
  • the partial or total loss of B2M function is assessed by comparing a cancer cell to a non-cancer cell from the same subject, wherein the non-cancer cell is from the same tissue from which the cancer cell was derived.
  • the partial or total loss of B2M function is assessed by comparing a cancer cell to a non-cancer cell from the same subject, wherein the non-cancer cell is not from the same tissue from which the cancer cell was derived. In some embodiments, the partial or total loss of B2M function is assessed by comparing a cancer cell to a non-cancer cell from a different subject. In some embodiments, the partial or total loss of B2M function is assessed by comparing a cancer cell to a non-cancer cell control.
  • the cancer cell is in a solid tumor that comprises cancer cells with normal B2M function. In some embodiments, the cancer cell is in a solid tumor in which 25% or more of the cancer cells have a partial or total loss in B2M function. In some embodiments, the cancer cell is in a solid tumor in which 50% or more of the cancer cells have a partial or total loss in B2M function. In some embodiments, the cancer cell is in a solid tumor in which 75% or more of the cancer cells have a partial or total loss in B2M function. In some embodiments, the cancer cell is in a solid tumor in which 95% or more of the cancer cells have a partial or total loss in B2M function.
  • the subject comprises a cell comprising a mutation in the B2M gene.
  • the mutation is a substitution, insertion, or deletion.
  • the B2M gene comprises a loss of heterozygosity (LOH).
  • the mutation is a frameshift mutation.
  • the mutation is a deletion mutation.
  • the frameshift mutation is in exon 1 of B2M.
  • the frameshift mutation results in a truncation of B2M.
  • the mutation is a complete or partial deletion ( e.g ., truncation) of B2M.
  • a deletion mutation is in exon 1 of B2M.
  • the frameshift mutation comprises p.Leul3fs and/or p.Serl4fs. In some embodiments, the frameshift mutation comprises V69Wfs*34, L15fs*41, L13P, L15fs*41, and/or p.S31* according to Middha et al. (2019) JCO Precis Oncol. (doi: 10.1200/PO.18.00321). In some embodiments, the mutation comprises a frameshift and/or deletion (e.g., truncation) mutation upstream of a kinase domain for JAK1 and/or JAK2.
  • deletion e.g., truncation
  • the subject has a reduced level of B2M protein as compared to a subject without a partial or total loss of B2M function.
  • the subject comprises a partial or total loss of beta-2 - microglobulin (B2M) function.
  • the subject comprises a partial loss of B2M function.
  • the subject comprises a total loss of B2M function.
  • the partial or total loss of B2M function may be assessed by comparing to a tissue sample from the same subject.
  • the partial or total loss of B2M function may be assessed by comparing a tissue sample from the tumor to a tissue sample from the same tissue from which the tumor sample was derived.
  • the solid tumor as a whole e.g ., as assessed in a biopsy taken from the solid tumor
  • the subject comprises (e.g., the partial or total loss of function results from) a mutation in the B2M gene.
  • certain cells within the tumor have a B2M loss of function. In some embodiment, certain cells within the tumor have a partial or total loss of B2M function while other cells in the tumor do not.
  • the subject has a reduced level of surface expressed major histocompatibility complex class I (MHC I) as compared to a control, optionally wherein the control is a non-cancerous sample from the same subject.
  • MHC I surface expressed major histocompatibility complex class I
  • a subject has a cancer cell comprising a reduced level of surface expressed MHC I.
  • the cancer cell has no surface expressed MHC I.
  • the reduced level of surface expressed MHC I is assessed by comparing a cancer cell to a non-cancer cell from the same subject, optionally wherein the non-cancer cell is from the same tissue from which the cancer cell was derived.
  • the cancer cell is in a solid tumor that comprises cancer cells with a normal level of surface expressed MHC I. In some embodiments, the cancer cell is in a solid tumor in which 25% or more of the cancer cells have a reduced level of surface expressed MHC I. In some embodiments, the cancer cell is in a solid tumor in which 50% or more of the cancer cells have a reduced level of surface expressed MHC I. In some
  • the cancer cell is in a solid tumor in which 75% or more of the cancer cells have a reduced level of surface expressed MHC I. In some embodiments, the cancer cell is in a solid tumor in which 95% or more of the cancer cells have a reduced level of surface expressed MHC I.
  • the solid tumor as a whole e.g, as assessed in a biopsy taken from the solid tumor
  • the RNAs/anti-PD-1 antibody combination provided herein is used in a method of treating an advanced-stage solid tumor cancer.
  • the RNAs/anti-PD-1 antibody combination provided herein is used in a method of treating an unresectable solid tumor cancer.
  • the RNAs/anti-PD-1 antibody combination provided herein is used in a method of treating a metastatic solid tumor cancer.
  • the cytokine RNA mixture is injected into one or more a solid tumor cancer within a lymph node.
  • the advanced- stage solid tumor cancer comprises a tumor that is suitable for direct intratumoral injection.
  • the advanced-stage solid tumor cancer is stage III, subsets of stage III, stage IV, or subsets of stage IV.
  • the cancer is melanoma.
  • the melanoma is stage MB, stage IIIC, or stage IV.
  • the cancer is cutaneous squamous cell carcinoma (CSCC).
  • the cancer is head and neck squamous cell carcinoma
  • the solid tumor cancer is melanoma, optionally wherein the melanoma is uveal melanoma or mucosal melanoma; and comprises superficial, subcutaneous and/or lymph node metastases amenable for intratumoral injection.
  • the solid tumor cancer is HNSCC and/or mucosal melanoma with only mucosal sites.
  • the solid tumor cancer is HNSCC.
  • the solid tumor cancer is uveal melanoma or mucosal melanoma.
  • the solid tumor cancer is uveal melanoma. In some embodiments, the solid tumor cancer is mucosal melanoma. In some embodiments, the RNAs are injected intratum orally only at mucosal sites of the solid tumor cancer, wherein the solid tumor cancer is HNSCC or mucosal melanoma.
  • the subject has failed a prior anti-programmed cell death 1 (PD-1) or anti-programmed cell death 1 ligand (PD-L1) therapy.
  • the subject has not been treated previously with an anti-PD-1 or anti-PD-Ll therapy.
  • the subject is without other treatment options.
  • the method may comprise reducing the size of a tumor or preventing cancer metastasis in a subject.
  • the subject has at least two tumor lesions or at least three tumor lesions. In some embodiments, the subject has two tumor lesions. In some embodiments, the subject has three tumor lesions.
  • the subject has measurable disease according to the Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 criteria as described herein.
  • RECIST Response Evaluation Criteria in Solid Tumors
  • the subject has a tumor that is suitable for direct intratumoral injection. In some embodiments, whether a tumor is suitable for direct
  • intratumoral injection may be based on the dose volume.
  • a tumor is suitable for direct intratumoral injection of a cytokine RNA mixture if it includes a cutaneous or subcutaneous lesion >0.5 cm in longest diameter or multiple injectable merging lesions which become confluent and have the longest diameter (sum of diameters of all involved target lesions) of >0.5 cm suitable for injection (z.e., not bleeding or weeping).
  • lymph nodes >1.5 cm that are suitable for ultrasonography (USG)-guided intratumoral injection and confirmed as metastatic disease are also suitable.
  • the tumor is uveal melanoma or mucosal melanoma.
  • the tumor is uveal melanoma or mucosal melanoma; and comprises superficial, subcutaneous and/or lymph node metastases amenable for intratumoral injection.
  • the subject is human. In some embodiments, the subject may have a life expectancy of more than 3 months, 4 months, 5 months or 6 months. In some embodiments, the subject has a life expectancy of more than 3 months. In some embodiments, the subject is at least 18 years of age.
  • methods for treating an advanced- stage melanoma, cutaneous squamous cell carcinoma (CSCC) or head and neck squamous cell carcinoma (HNSCC) comprising administering to a subject having an advanced- stage melanoma RNA encoding an IL-12sc protein, RNA encoding an IL-15 sushi protein, RNA encoding an IFNa protein, and RNA encoding a GM-CSF protein and an anti -PD- 1 antibody.
  • the subject is at least 18 years of age; (b) the subject has failed prior anti -PD 1 or anti-PD-Ll therapies; (c) the subject has a minimum of 2 lesions; and (d) the melanoma, CSCC, or HNSCC comprises a tumor that is suitable for direct intratumoral injection.
  • the subject has measurable disease according to the Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 criteria. In some embodiments, the subject has a life expectancy of more than 3 months.
  • the solid tumor is an epithelial tumor, prostate tumor, ovarian tumor, renal cell tumor, gastrointestinal tract tumor, hepatic tumor, colorectal tumor, tumor with vasculature, mesothelioma tumor, pancreatic tumor, breast tumor, sarcoma tumor, lung tumor, colon tumor, melanoma tumor, small cell lung tumor, neuroblastoma tumor, testicular tumor, carcinoma tumor, adenocarcinoma tumor, seminoma tumor, retinoblastoma, cutaneous squamous cell carcinoma (CSCC), squamous cell carcinoma for the head and neck (HNSCC), head and neck cancer, or osteosarcoma tumor.
  • CSCC cutaneous squamous cell carcinoma
  • HNSCC head and neck cancer
  • osteosarcoma tumor cutaneous squamous cell carcinoma
  • the solid tumor comprises a primary tumor of any size. In some embodiments, tumor thickness measurements are reported rounded to the nearest 0.1 mm. In some embodiments, the solid tumor comprises a primary tumor having ⁇ 1.0 mm in thickness. In some embodiments, the solid tumor comprises a primary tumor having 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 mm in thickness. In some embodiments, the solid tumor comprises a primary tumor having ⁇ 0.8 mm (or less than 0.8 mm) in thickness without ulceration. In some embodiments, the solid tumor comprises a primary tumor having ⁇ 0.8 mm (or less than 0.8 mm) in thickness with ulceration.
  • the solid tumor comprises a primary tumor having from 0.8 to 1.0 mm in thickness. In some embodiments, the solid tumor comprises a primary tumor having 0.8, 0.9, or 1.0 mm in thickness. In some embodiments, the solid tumor comprises a primary tumor having from 0.8 to 1.0 mm in thickness without or with ulceration. In some embodiments, the solid tumor comprises a primary tumor having >1.0 - 2.0 mm in thickness. In some embodiments, the solid tumor comprises a primary tumor having 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 mm in thickness. In some embodiments, the solid tumor comprises a primary tumor having >1.0 - 2.0 mm in thickness without or with ulceration.
  • the solid tumor comprises a primary tumor having >2.0 - 4.0 mm in thickness. In some embodiments, the solid tumor comprises a primary tumor having 3.0 - 4.0 mm in thickness. In some embodiments, the solid tumor comprises a primary tumor having 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or 4.0 mm in thickness. In some embodiments, the solid tumor comprises a primary tumor having >2.0 - 4.0 mm in thickness without or with ulceration. In some embodiments, the solid tumor comprises a primary tumor having >4.0 mm in thickness.
  • the solid tumor comprises a primary tumor having 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 7.0, 8.0, 9.0 or 10.0 mm in thickness.
  • the solid tumor comprises a primary tumor having >4.0 mm in thickness without or with ulceration.
  • the thickness is at the thickest (i.e., greatest) dimension of the tumor.
  • the tumor is a skin cancer tumor and the thickness is from the skin surface to the deepest part of the tumor ( e.g ., the thickness is not the lateral spread of the tumor).
  • the tumor is a skin metastasis of a cancer other than a skin cancer, and the thickness of the tumor is from the skin surface to the deepest part of the tumor (e.g., the thickness is not the lateral spread of the tumor).
  • the solid tumor is a melanoma solid tumor.
  • the melanoma comprises a primary tumor of any size.
  • tumor thickness measurements are reported rounded to the nearest 0.1 mm.
  • the melanoma comprises a primary tumor having ⁇ 1.0 mm in thickness. In some embodiments, the melanoma comprises a primary tumor having 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 mm in thickness. In some embodiments, the melanoma comprises a primary tumor having ⁇ 0.8 mm (or less than 0.8 mm) in thickness without ulceration. In some embodiments, the melanoma comprises a primary tumor having ⁇ 0.8 mm (or less than 0.8 mm) in thickness with ulceration. In some embodiments, the melanoma comprises a primary tumor having from 0.8 to 1.0 mm in thickness.
  • the melanoma comprises a primary tumor having 0.8, 0.9, or 1.0 mm in thickness. In some embodiments, the melanoma comprises a primary tumor having from 0.8 to 1.0 mm in thickness without or with ulceration.
  • the melanoma comprises a primary tumor having >1.0 - 2.0 mm in thickness. In some embodiments, the melanoma comprises a primary tumor having 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 mm in thickness. In some embodiments, the melanoma comprises a primary tumor having >1.0 - 2.0 mm in thickness without or with ulceration. In some embodiments, the melanoma comprises a primary tumor having >2.0 - 4.0 mm in thickness. In some embodiments, the melanoma comprises a primary tumor having 3.0 - 4.0 mm in thickness.
  • the melanoma comprises a primary tumor having 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or 4.0 mm in thickness.
  • the melanoma comprises a primary tumor having >2.0 - 4.0 mm in thickness without or with ulceration.
  • the melanoma comprises a primary tumor having >4.0 mm in thickness.
  • the melanoma comprises a primary tumor having 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 7.0, 8.0, 9.0 or 10.0 mm in thickness.
  • the melanoma comprises a primary tumor having >4.0 mm in thickness without or with ulceration.
  • the thickness is from the skin surface to the deepest part of the tumor (the thickness is not the lateral spread of the tumor).
  • the melanoma comprises one tumor-involved regional lymph node or any number of in-transit, satellite, and/or microsatellite metastases with no tumor-involved nodes. In some embodiments, the melanoma comprises one clinically occult tumor-involved regional lymph node. In some embodiments, the melanoma comprises one clinically detectable tumor-involved regional lymph node. In some embodiments, the melanoma comprises any number of in-transit, satellite, and/or microsatellite metastases with no tumor- involved nodes.
  • the melanoma comprises two or three tumor-involved regional lymph nodes or any number of in-transit, satellite, and/or microsatellite metastases with no tumor-involved nodes. In some embodiments, the melanoma comprises two or three clinically occult tumor-involved regional lymph nodes. In some embodiments, the melanoma comprises two or three tumor-involved regional lymph nodes, at least one of which is clinically detectable. In some embodiments, the melanoma comprises two or three tumor-involved regional lymph nodes, one of which is clinically occult or clinically detectable and with presence of in-transit, satellite, and/or microsatellite metastases.
  • the melanoma comprises any number of in-transit, satellite, and/or microsatellite metastases with one tumor-involved node. In some embodiments, the melanoma comprises four or more tumor- involved regional lymph nodes or any number of in-transit, satellite, and/or microsatellite metastases with two or more tumor-involved nodes or any number of matted nodes without or with in-transit, satellite, and/or microsatellite metastases. In some embodiments, the melanoma comprises four or more clinically occult tumor-involved regional lymph nodes.
  • the melanoma comprises four or more clinically occult tumor-involved regional lymph nodes, at least one of which is clinically detectable or with presence of any number of matted nodes. In some embodiments, the melanoma comprises two or three tumor-involved regional lymph nodes, one of which is clinically occult or clinically detectable. In some embodiments, the melanoma comprises four or more clinically occult tumor-involved regional lymph nodes, two or more of which are clinically occult or clinically detectable and/or with presence of any number of matted nodes, and with presence of in-transit, satellite, and/or microsatellite metastases.
  • a. comprises a primary tumor of any size
  • b. comprises one or more tumor-involved regional lymph nodes; or in-transit, satellite, and/or microsatellite metastases with no tumor-involved regional lymph nodes; and
  • c. comprises no detectable distant metastasis.
  • the melanoma has a detectable distant metastasis.
  • a. comprises a primary tumor having ⁇ 0.8 mm in thickness without ulceration; or a primary tumor having from 0.8 to 1.0 mm in thickness and a primary tumor less than 0.8 mm in thickness with ulceration; or a primary tumor having >1.0-2.0 mm in thickness without ulceration;
  • b. comprises one or two or three clinically occult tumor-involved regional lymph nodes
  • c. comprises no detectable distant metastasis.
  • a. comprises a primary tumor having ⁇ 0.8 mm in thickness without ulceration; or a primary tumor having from 0.8 to 1.0 mm in thickness and a primary tumor less than 0.8 mm in thickness with ulceration; or a primary tumor having >1.0-2.0 mm in thickness without ulceration;
  • b. comprises one clinically detectable tumor-involved regional lymph node; or no tumor-involved regional lymph node with presence of in-transit, satellite, and/or microsatellite metastases; or two or three tumor-involved regional lymph nodes, at least one of which is clinically detectable; and
  • c. comprises no detectable distant metastasis.
  • the melanoma a comprises a primary tumor having >1.0-2.0 mm in thickness with ulceration; or a primary tumor having >2.0-4.0 mm in thickness without ulceration; b. comprises one clinically detectable or clinically occult tumor-involved regional lymph node; or none or one tumor-involved regional lymph nodes with in-transit, satellite, and/or microsatellite metastases; and
  • c. comprises no detectable distant metastasis.
  • a. comprises one clinically detectable tumor-involved regional lymph node; or no tumor-involved regional lymph nodes with presence of in-transit, satellite, and/or microsatellite metastases; and
  • b. comprises no detectable distant metastasis.
  • the melanoma has no detectable distant metastasis; and comprises
  • tumor-involved regional lymph node b. one clinically occult or detectable tumor-involved regional lymph node with presence of in-transit, satellite, and/or microsatellite metastases; c. four or more tumor-involved regional lymph nodes, at least one of which is
  • the melanoma comprises a primary tumor having ⁇ 0.8 mm or >1.0-2.0 or >2.0-4.0 mm in thickness without ulceration; comprises no detectable distant metastasis; and comprises:
  • the melanoma a. one clinically occult or clinically detected tumor-involved regional lymph nodes with presence of in-transit, satellite, and/or microsatellite metastases; or b. four or more tumor-involved regional lymph nodes; or one or more in-transit, satellite, and/or microsatellite metastases with two or more tumor-involved nodes; or one or more matted nodes without or with in-transit, satellite, and/or microsatellite metastases.
  • the melanoma a. one clinically occult or clinically detected tumor-involved regional lymph nodes with presence of in-transit, satellite, and/or microsatellite metastases.
  • a comprises a primary tumor having >2.0-4.0 mm in thickness with ulceration or a primary tumor having >4.0 mm in thickness without ulceration;
  • b comprises one or more tumor-involved regional lymph nodes; or one or more in transit, satellite, and/or microsatellite metastases optionally with one or more tumor-involved regional lymph nodes; or one or more matted nodes without or with in-transit, satellite, and/or microsatellite metastases; and
  • c. comprises no detectable distant metastasis.
  • a. comprises a primary tumor in >4.0 mm in thickness without ulceration;
  • b. comprises one or two or three tumor-involved regional lymph nodes; or one or more in-transit, satellite, and/or microsatellite metastases with no or one tumor- involved regional lymph nodes; and
  • c. comprises no detectable distant metastasis.
  • a. comprises a primary tumor >4.0 mm in thickness with ulceration
  • b. comprises four or more tumor-involved regional lymph nodes; or one or more in transit, satellite, and/or microsatellite metastases with two or more tumor- involved regional lymph nodes, or one or more matted nodes without or with in transit, satellite, and/or microsatellite metastases; and
  • c. comprises no detectable distant metastasis.
  • the cutaneous squamous cell carcinoma (CSCC) or squamous cell carcinoma for the head and neck (HNSCC) comprises a tumor of any size. In some embodiments, the CSCC or HNSCC comprises no identified tumor. In some
  • the CSCC or HNSCC comprises a tumor that is 2 cm or smaller in its greatest dimension. In some embodiments, the CSCC or HNSCC comprises a tumor larger than 2 cm but not larger than 4 cm in its greatest dimension. In some embodiments, the CSCC or HNSCC comprises a tumor that is larger than 4 cm in greatest dimension or has minimal erosion of the bone or perineural invasion or deep invasion. In some embodiments, the CSCC or HNSCC comprises a tumor with extensive cortical or medullary bone involvement or invasion of the base of the cranium or invasion through the foramen of the base of the cranium.
  • the cutaneous squamous cell carcinoma (CSCC) or squamous cell carcinoma for the head and neck (HNSCC) comprises no regional lymph node metastasis.
  • the CSCC or HNSCC comprises metastasis in a single ipsilateral lymph node, is 3 cm or smaller in greatest dimension, and is ENE-negative.
  • the CSCC or HNSCC comprises metastasis in a single ipsilateral lymph node larger than 3 cm but not larger than 6 cm in greatest dimension and ENE-negative.
  • the CSCC or HNSCC comprises metastases in multiple ipsilateral lymph nodes, none larger than 6 cm in their greatest dimension and is ENE-negative.
  • the CSCC or HNSCC comprises metastasis in bilateral or contralateral lymph nodes, none larger than 6 cm in greatest dimension, and is ENE-negative. In some embodiments, the CSCC or HNSCC comprises metastasis in a lymph node larger than 6 cm in its greatest dimension and is ENE-negative; or metastasis in any lymph nodes and ENE-negative. In some embodiments, the cutaneous squamous cell carcinoma (CSCC) or squamous cell carcinoma for the head and neck (HNSCC):
  • a. comprises a tumor larger than 4 cm in greatest dimension or has minimal erosion of the bone or perineural invasion or deep invasion;
  • c. comprises no detectable distant metastasis.
  • the cutaneous squamous cell carcinoma (CSCC) or squamous cell carcinoma for the head and neck (HNSCC) comprises:
  • metastasis in a single ipsilateral lymph node 3 cm or smaller in its greatest dimension and is ENE-negative;
  • the cutaneous squamous cell carcinoma (CSCC) or squamous cell carcinoma for the head and neck (HNSCC) comprises:
  • a tumor larger than 2 cm but not larger than 4 cm in its greatest dimension a tumor larger than 2 cm but not larger than 4 cm in its greatest dimension
  • metastasis in a single ipsilateral lymph node, 3 cm or smaller in its greatest dimension and is ENE-negative a tumor larger than 2 cm but not larger than 4 cm in its greatest dimension
  • CSCC cutaneous squamous cell carcinoma
  • HNSCC squamous cell carcinoma for the head and neck
  • a. comprises: i. a tumor that is 2 cm or smaller in its greatest dimension; or
  • a tumor larger than 2 cm but not larger than 4 cm in its greatest dimension ii. a tumor larger than 2 cm but not larger than 4 cm in its greatest dimension; or iii. a tumor larger than 4 cm in its greatest dimension or minimal erosion of the bone or perineural invasion or deep invasion; and
  • n. metastases in multiple ipsilateral lymph nodes none larger than 6 cm in its greatest dimension and is ENE-negative; or
  • c. comprises no detectable distant metastasis.
  • CSCC cutaneous squamous cell carcinoma
  • HNSCC squamous cell carcinoma for the head and neck
  • a. comprises
  • a tumor larger than 2 cm but not larger than 4 cm in its greatest dimension ii. a tumor larger than 4 cm in greatest dimension or minimal erosion of the bone or perineural invasion or deep invasion; or
  • CSCC cutaneous squamous cell carcinoma
  • HNSCC squamous cell carcinoma for the head and neck
  • a. comprises tumor with extensive cortical or medullary b one involvement or invasion of the base of the cranium or invasion through the foramen of the base of the cranium;
  • CSCC cutaneous squamous cell carcinoma
  • HNSCC squamous cell carcinoma for the head and neck
  • a. comprises tumor with extensive cortical or medullary bone involvement or
  • b. comprises no detectable distant metastasis.
  • CSCC cutaneous squamous cell carcinoma
  • HNSCC squamous cell carcinoma for the head and neck
  • a. comprises
  • a tumor 2 cm or smaller in greatest dimension i. a tumor 2 cm or smaller in greatest dimension; or ii. a tumor larger than 2 cm but not larger than 4 cm in greatest dimension; or iii. a tumor larger than 4 cm in greatest dimension or minimal erosion of the bone or perineural invasion or deep invasion; or
  • the cutaneous squamous cell carcinoma (CSCC) or squamous cell carcinoma for the head and neck (HNSCC) comprises no detectable distant metastasis.
  • the therapeutically effective amount of the RNAs results in one or more of: (a) a reduction in the severity or duration of a symptom of cancer; (b) inhibition of tumor growth, or an increase in tumor necrosis, tumor shrinkage and/or tumor disappearance; (c) delay in tumor growth and/or development; (d) inhibited or retarded or stopped tumor metastasis; (e) prevention or delay of recurrence of tumor growth; (f) increase in survival of a subject; and/or (g) a reduction in the use or need for conventional anticancer therapy (e.g ., reduced or eliminated use of chemotherapeutic or cytotoxic agents), optionally as compared to an untreated subject or a subject administered only 1, 2, or 3 of the RNAs in the RNA mixture.
  • This description and exemplary embodiments should not be taken as limiting.
  • the cytokine RNA mixture as defined above, may be also referred as“the mixture,”“the cytokine mixture,”“the composition,” or“the drug” interchangeably.
  • Example 1 Dose escalation and dose expansion of the cytokine RNA mixture as
  • Enrollment of up to 192 participants is planned, when the cytokine RNA mixture is administered in combination with cemiplimab, depending on the investigated dose levels during the escalation phase and the completed stages for each cohort during the expansion phase. Together, enrollment of up to 264 participants is planned.
  • Dose escalation phase (monotherapy): There is no formal sample size calculation in the dose escalation phase.
  • the cytokine RNA mixture is administered to patients with advanced solid tumors who have failed a prior anti- PD-1 or anti-PD-Ll based therapy, and/or patients without other treatment options for those indications in which anti -PD-1 is not routinely used.
  • Up to 38 dose limiting toxi cities (DLT)-evaluable participants enroll in the dose escalation phase with expected assessment of about 8 dose levels. The actual sample size varies depending on DLTs observed and number of dose levels actually explored.
  • Dose expansion phase (monotherapy): A Simon’s two-stage design is used in the expansion phase and approximately 34 participants with advanced melanoma who failed prior anti-PD-l/anti-PD-Ll therapies enroll. After the first 16 treated participants, there is an interim analysis, and if response is observed in at least 2 participants, accrual continues to the full sample size of 34 participants.
  • Dose escalation phase (combination therapy): The actual sample size in the dose escalation of the cytokine RNA mixture in combination with cemiplimab varies depending on DLTs observed and number of dose levels actually explored (approximately 18 to 36 DLT- evaluable participants).
  • Dose expansion phase (combination therapy): A Simon’s two stage design is used in expansion phase of the cytokine RNA mixture in combination with cemiplimab and approximately 156 participants with advanced melanoma, CSCC, or HNSCC are enrolled in four cohorts. An interim analysis is performed at the end of Stage 1 of the Simon’s two-stage design for each cohort (26 patients for Cohort A, 14 patients for Cohort B, 10 patients for Cohort C, 26 patients for Cohort D). Enrollment in all cohorts (A, B, C, and D) is performed in parallel.
  • Intervention groups and duration The duration of the study for a participant includes a period for screening of up to 28 days. Once successfully screened, participants may receive study intervention until disease progression, unacceptable AE, participant’s decision to stop the treatment, or for a maximum of 1 year if no disease progression occurs. Continuation of the cytokine RNA mixture as a single agent and in combination with cemiplimab will be considered beyond 1 year by the study committee on a case by case basis for those participants that clearly continue to derive clinical benefit in a safe manner with reasonable toxicity. After discontinuing study intervention, participants return to the study site approximately 30 days after the last IMP administration or before the participant receives another anticancer therapy, whichever is earlier, for end-of-treatment assessments. If the participant discontinues study intervention for reasons other than progression, follow-up visits are performed every 3 months until disease progression, initiation of another anticancer treatment, or death (whichever comes first).
  • the expected duration of treatment for participants who benefit from the cytokine RNA mixture and/or cemiplimab may vary, based on progression date; but median expected duration of study per participant is estimated as 9 months (1 month for screening, 5 months for treatment, and 3 months for end of treatment follow-up) and 12 months in combination therapy (1 month for screening, 8 months for treatment, and 3 months for end of treatment follow-up).
  • the cytokine RNA mixture is administered intratumorally once per week in a 4-week cycle (i.e., four doses every 28 days).
  • the cytokine RNA mixture is administered intratumorally once per week with cemiplimab administered intravenously on Day 1 of a 3-week cycle (i.e., three doses of the cytokine RNA mixture and one dose of cemiplimab every 21 days).
  • Intratumoral injection is to continue weekly until the second tumor assessment, at which time a change of the dose interval of the cytokine RNA mixture to twice or once a month (in monotherapy) or every three weeks (in combination therapy) may be considered; a more flexible dose interval of the cytokine RNA mixture administration may also be considered depending on available data.
  • Dose omissions or dose delay may occur throughout the study; the occurrence of dose limiting toxicities (DLTs) determines the need for these modifications. Participants who experience a DLT have their treatment stopped (in either monotherapy or combination therapy), and are followed until resolution to Grade ⁇ 1 or baseline status. The study intervention is definitively discontinued in case a DLT is observed during the DLT observation period. If an AE meets the DLT criteria and occurs after the DLT observation period, a benefit-risk assessment is made on a case by case basis to decide whether to continue therapy.
  • DLTs dose limiting toxicities
  • the participant may resume therapy with a new cycle of treatment at the same or a lower dose level; no dose re-escalation is allowed for such re-dosed participants at a lower dose level. If the participant experiences the same AE leading to a second dose omission for 2 weeks (i.e., 2 dose omissions), then the participant may be permanently discontinued.
  • cemiplimab Participants receiving cemiplimab remain on the assigned dosage throughout the course of study treatment (350 mg Q3W), and no dose modifications are allowed for cemiplimab if not considered as a mandatory requirement due to safety profile; however, treatment cycle delay or an omission of cemiplimab dose are permitted if needed due to toxicity. If a participant has a cemiplimab infusion-related allergic drug reaction that leads to the termination of cemiplimab treatment, the participant may continue the cytokine RNA mixture treatment at the assigned dose level as monotherapy.
  • Dose regimen the cytokine RNA mixture is administered at assigned dose levels once a week, 4 injections within a 28-day cycle.
  • the cemiplimab drug product is presented as a concentrated solution (50 mg/mL) containing 10 mM histidine, 5% (w/v) sucrose, 1.5% (w/v) L-proline, and 0.2% (w/v) polysorbate 80, at pH 6.0, and is provided in 10 mL single-use vials filled to one of two different withdrawable amounts:
  • cemiplimab is administered intravenously at the fixed recommended dose of 350 mg Q3W, followed by the cytokine RNA mixture administered intratumorally weekly. On days of treatment with both cemiplimab and the cytokine RNA mixture, cemiplimab is administered first followed by the cytokine RNA mixture (on the same day).
  • Noninvestigational medicinal products(s) No pre-defmed premedication is administered.
  • Dose escalation In the dose escalation phase, DLTs are summarized by dose level. Details of DLTs are provided by participant. The treatment-emergent AEs/SAEs and laboratory abnormalities during the on-treatment period are summarized using descriptive statistics by dose level.
  • Dose expansion (monotherapy and combination therapy): Objective response rate (ORR) per RECIST 1.1 are summarized with descriptive statistics. A 90% two-sided confidence interval is computed using Clopper- Pearson method. The statistical inference is based on the hypothesis and alpha level defined in the sample size calculation section.
  • Dose escalation (monotherapy and combination therapy): Concentration and PK parameters of the cytokines encoded by the cytokine RNA mixture and of cemiplimab are summarized with descriptive statistics during cycles in which PK is assessed. Anti-drug antibodies (AD As) against the cytokines encoded by the cytokine RNA mixture and AD As against cemiplimab are descriptively summarized.
  • Dose expansion (monotherapy and combination therapy): The treatment-emergent AEs/SAEs and laboratory abnormalities during the on-treatment period are summarized using descriptive statistics. DoR and PFS per RECIST 1.1 and iRECIST are summarized using the Kaplan-Meier method.
  • ORR per RECIST 1.1 A similar analysis as ORR per RECIST 1.1 is provided for DCR per RECIST 1.1 and iRECIST, and the ORR per iRECIST.
  • PK concentration and parameters of the cytokines encoded by the cytokine mixture and of cemiplimab are summarized with descriptive statistics during cycles in which PK is assessed.
  • Fig. 1 A shows a graphic of the overall design of the treatments (top:
  • FIG. IB and 1C shows a graphic of the treatment scheduling for monotherapy and for combination therapy respectively.
  • the dose escalation phase in monotherapy aims to determine the MTD or MAD of the cytokine RNA mixture administered weekly as monotherapy. (Fig. 1 A,“a”).
  • Stage IIIB-C or Stage IV Melanoma after failure of anti-PD-1 or anti-PD-Ll are eligible.
  • the number of included participants is up to 34 participants if at least two responses observed in first 16 treated participants. (Fig. 1 A,“b”).
  • Fig. 1 A,“c”) the occurrence of toxicities observed in Cycle 1 is assessed on one participant per cohort.
  • the dose escalation phase in combination therapy aims to determine the MTD or MAD of the cytokine RNA mixture administered weekly in combination with cemiplimab administered IV once every 3 weeks.
  • the dose escalation of the cytokine RNA mixture in combination with cemiplimab is initiated during the ongoing monotherapy dose escalation, once a dose level has been demonstrated to be safe and tolerable in monotherapy (based on a 28-day DLT observation period); once signs of PK, PDy, and/or clinical response (systemic and/or local) have been shown.
  • the DLs explored in combination are the same as in monotherapy. If the site participates in both of the dose escalation phases (i.e., dose escalation in both monotherapy and combination therapy parts of the study), the dose escalation phase in the monotherapy part of the study is prioritized. (Fig.
  • Cohort A Melanoma after anti-PD-l/PD-Ll failure
  • Cohort B Melanoma anti-PD-l/PD LI naive
  • Cohort C CSCC anti-PD-l/PD LI naive
  • Cohort D HNSCC anti-PD-l/PD LI naive.
  • Tables 2 and 3 show the Schedule of Activities (SOA) for monotherapy with Table 2 showing the treatment flowchart and Table 3 showing the PK and PDy flowchart for the dose escalation and expansion phases.
  • Tables 4 and 5 show the Schedule of Activities (SOA) for combination therapy with Table 4 showing the treatment flowchart and Table 5 showing the PK and PDy flowchart for the dose escalation and expansion phases.
  • Table 2 Schedule of Activities (SO A) and Treatment Flowchart- Monotherapy
  • a cycle is 28 days, with the cytokine RNA mixture administered intratumorally every week as monotherapy.
  • stage at diagnosis and at study entry includes stage at diagnosis and at study entry, and previous anti-tumor therapy (type, duration, reason for discontinuation and response to the therapy).
  • type includes stage at diagnosis and at study entry, and previous anti-tumor therapy (type, duration, reason for discontinuation and response to the therapy).
  • specific mutations depending on tumor type.
  • d Body weight is measured prior to treatment on the first day of each cycle.
  • e Height is measured during baseline only.
  • Vital signs include: temperature, blood pressure, heart rate, respiration rate. Vital signs must be checked every 6 hours during each 24 hour inpatient hospitalization period during C1D1 at each new dose level while participants are monitored to assess for acute toxicities.
  • g Physical examination includes: examination of major body systems including cardiovascular system, digestive system, central nervous system, respiratory system, and hematopoietic system (hepatomegaly, splenomegaly, lymphadenopathy), and skin. Signs and symptoms are reported in the eCRF as AEs only if they are still present at the time of first IMP administration.
  • color digital photographs are mandatory starting at DL4 of mono escalation, starting from first DL in combo escalation and during expansion phase. Digital photographs are mandatory at screening prior to first dose of cytokine RNA mixture and at the time of radiographic tumor assessment from superficial and/or visible subcutaneous injected lesions to document overall disease status and to document responses. In addition, ad hoc color digital photographs must be taken in between screening and tumor assessment windows to capture other cytokine RNA mixture potentially induced changes such as skin redness and/or edema. All collected by the clinical site must be systematically shared with the Sponsor for review as per study reference manual.
  • j Blood hematology Hemoglobin, hematocrit, WBC with differential (including absolute neutrophil count [ANC]), platelet count. These tests are done before each IMP administration (-1 day window is acceptable). If Grade 4 neutropenia, assess ANC every 2-3 days until ANC >0.5 x 10 9 /L, then weekly until recovery. The Cycle 1 Day 1 assessment is done within 2 days of IMP administration, if abnormal at baseline.
  • k Coagulation activated partial thromboplastin time (aPTT), PT, international normalized ration (INR), fibrinogen (and D-dimer at Screening).
  • the Cycle 1 Day 1 assessment is done within 2 days of IMP administration, if abnormal at baseline.
  • Liver function tests AST, ALT, total bilirubin, direct bilirubin, alkaline phosphatase (ALP). Renal function tests: Urea or BUN & creatinine, and determination of estimated CrCL when required (if creatinine between 1.0 and 1.5x ULN).
  • Electrolytes Sodium, potassium, total calcium, phosphorus, chloride, magnesium and bicarbonate. Others: glucose, lactate dehydrogenase (LDH), albumin, total proteins, and amylase.
  • LDH lactate dehydrogenase
  • the liver function tests, renal function tests, electrolytes glucose, LDH, albumin and total proteins are performed before IMP administration (-1 day window is acceptable), unless clinically indicated. In case of Grade >3 liver function abnormal tests, additional tests are repeated every 2-3 days until recovery to baseline value.
  • the Cycle 1 Day 1 serum chemistry assessment is done within 2 days of IMP administration, if abnormal at baseline.
  • Serum C-reactive protein (CRP), ferritin, and secondary plasma cytokines are be collected at the specified time points and in case of occurrence of CRS Grade >2 symptoms.
  • Serum CRP and Ferritin samples are collected just before each study intervention (Dl) and at 24 hr (D2) during Cycle 1 (for each Week, 1 - 4) and during Cycle 3 Week 1.
  • Dl study intervention
  • D2 24 hr
  • C2 secondary plasma cytokines
  • Samples are collected at pre-dose and 6 and 24 horns after the cytokine RNA mixture administration at Cycle 1, Weeks 1 and 2, and Cycle 3 Week 1; at EOT; and in case of Grade >2 symptoms of CRS.
  • n 12-lead ECG to be done at screening and pretreatment at Cycle 1 Day 1, Cycle 3 Day 1, Cycle 7 Day 1, and EOT, and when clinically indicated.
  • FDG PET only applicable for patients with lymphoma as per Lugano classification to be performed within 28 days of IMP administration (-7 days), and approximately every 12 weeks ( ⁇ 7 days) to confirm CR and PD and as clinically indicated.
  • q Urinalysis Dipstick (qualitative) tests on morning spot by dipstick are performed at baseline and before each IMP administration and at EOT. Quantitative urinalysis for leukocytes and red blood cells on morning spot urine are performed at baseline, at uneven cycles, at the end of treatment, and in case of abnormality in the dipstick test (qualitative). In case of proteinuria >++ (dipstick), proteinuria quantification by proteinuria/24 hr urine collection is performed.
  • Urine biomarker kidney injury molecule-1 (KIM-1), urinary microalbumin, and urinary creatinine (in spot urine) are assessed at pre-dose on Cycle 1 Day 1 (within 7 days beforehand is acceptable), 24 hr after the first IMP administration, and pre-dose on day 8 after the first IMP administration.
  • KIM-1 kidney injury molecule-1
  • urinary microalbumin urinary microalbumin
  • urinary creatinine in spot urine
  • Ophthalmologic exam including Schirmer’s test is performed at baseline and in case of ocular symptoms during therapy. Ocular and visual symptoms are assessed on Day 1 of each Cycle.
  • Adverse Event assessment The period of observation for collection of adverse events extends from the signature of the Informed Consent Form (ICF) until 30 days after the last administration of the study drug. Serious adverse events are assessed and reported as described in the protocol. After the EOT visit, ongoing SAEs and AESIs, related AEs, and new related AEs are to be followed up to stabilization, recovery, or initiation of further therapy.
  • ICF Informed Consent Form
  • Concomitant medications are recorded from 14 days prior to the initial dose of study drug until 30 days after the last administration o study drug, resolution of ongoing study -drug related adverse events, or when another anticancer therapy is received
  • RNA mixture can be administered with a window of +/- 1 days during Cycle 1 and with a window of +/- 3 days starting from Cycle 2.
  • Tumor assessment CT-scan or magnetic resonance imaging (MRI) and any other exams as clinically indicated are performed to assess disease status at baseline (within 28 days of IMP administration +/- 7 days), every 8 weeks following IMP administration (-/+ 7 days) up to Week 24, then every 12 weeks (-/+ 7 days) and at the end of study intervention, except if already done at last cycle. Patients who discontinued study intervention without progressive disease are followed every 12 weeks until the documented progressive disease. Tumor assessment is repeated to confirm a partial or complete response as well as progressive disease (at least 4 weeks after initial documented response).
  • radiological tumor assessment of abdomen and thorax are performed at 24 weeks, if there is no clinical sign of metastatic disease, and at EOT if not already done at last cycle.
  • Intermittent ultrasonography (USG) or clinically indicated assessment can be considered in case of clinical signs or laboratory abnormalities, mainly liver function tests, to exclude potential metastatic disease
  • a Blood samples for PK are collected for evaluation of target expression of the cytokine RNA mixture -encoded cytokines in all enrolled participants on Cycle 1 Week 1 at pre-dose and 1, 2, 6, 24, 48, and 96 or 120 hours after IMP administration.
  • Cycle 1 Week 2 in the dose escalation phase samples are collected at pre-dose and 2, 6, 24, and 48 hours post dosing; in the dose expansion phase, Cycle 1 Week 2 sampling occurs only at pre-dose, 6, and 24 hours post dosing.
  • Cycle 1 Week 3 and subsequent Cycles see footnote ⁇ . Samples are also collected right before the tumor biopsy, at EOT and the first follow up visit. Further information is detailed in the study laboratory manual. No PK samples are collected following the second study cut-off date (see herein).
  • Cycle 3 Week 1 ie, week 9 from first administration
  • the schedule for Cycle 1 Week 1 is repeated.
  • PK sampling is to occur at 0 and 6 hours at Week 1 of every odd-numbered cycle.
  • PK samples of odd-numbered cycles can be omitted by notification of the Sponsor, if available data are considered sufficient
  • Blood sample for immune assessment and circulating factors Blood samples are collected at pre-dose, 6, and 24 hr of Cycle 1 Weeks 1 and 2, at EOT, and FU in all participants to assess systemic immune modulations including IFNy and IP10. Further information us be detailed in the study laboratory manual.
  • Cycle 3 Week 1 the sampling schedule for Cycle 1 Week 1 is repeated for immune assessment and circulating factors. Beyond Cycle 3, PDy sampling is to occur at 0 and 6 hrs at Week 1 of every odd-numbered Cycle. No sampling of blood for PDy cytokines occurs during even-numbered cycles during the monotherapy part of the study.
  • e Blood for genetic analysis is used to establish the germline DNA sequence and HLA typing.
  • f Blood samples (leukapheresis or 80 mL of blood) are collected pre-dose Cycle 1 Week 1, pre-dose Cycle 2 Week 2 (ie, 5 weeks post-dose on Cycle 1), and at EOT for the analysis of antigen specific T-cell. This analysis will occur only for participants with melanoma in the monotherapy escalation phase and for all participants (melanoma) in the monotherapy expansion phase.
  • Tumor biopsy for immune assessment biopsies are collected during the screening period (before IMP administration on Cycle 1 Day 1), between Weeks 5 and 8, and at Cycle 6 or at disease progression (whichever occurs first), to assess immune modulations.
  • Tumor transcriptomics RNA sequencing
  • genomics genomics
  • neo-antigens and TIL isolation
  • TIL isolation may also be performed upon sample availability (see herein).
  • a single tumor core biopsy performed between Weeks 5-8 is dedicated for TILs isolation. This is applied to a limited number (aiming no more than 10 patients with successful TILs isolation) of selected melanoma patients (expansion for monotherapy and only in Cohort A of combination therapy expansion). This will not be an additional biopsy, but instead the sample dedicated for genomic assessment will be used for TILs isolation (handled under special conditions-not formalin fixed).
  • This kind of sample and testing is applied to patients with clinical signs of response to treatment (tumor size reduction and/or redness at the tumor site) as determined by the treating investigator.
  • Plasma samples to monitor development of antibodies to the cytokine RNA mixture -encoded cytokines are collected pre-dose Day 1 for Cycles 1, 3, 6, 9, 12 and/or EOT, and at FU (Day 90 after last IMP administration). Additional collections beyond these timepoints are every 3 months if the participant continues on study for follow-up visits. No ADA samples are collected following the second cut-off date.
  • Table 4 Schedule of Activities (SOA) and Treatment Flowchart- Combination Therapy
  • Tumor biopsy are collected for immunohistochemistry, genomic, RNA-sequencing, and neo-antigen analyses.
  • a cycle In combination with Cemiplimab a cycle is 21 days, with the cytokine RNA mixture administered intratumorally weekly and Cemiplimab administered at the fixed dose of 350 mg
  • c Demography Includes age, gender, and race.
  • Medical/Surgical History Includes relevant history of previous pathologies and surgeries.
  • Disease History Includes stage at diagnosis and at study entry, and previous anti-tumor therapy (type, duration, reason for discontinuation and response to the therapy). In addition, specific mutations depending on tumor type.
  • e Height is measured during baseline only.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Epidemiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biophysics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Endocrinology (AREA)
  • Mycology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

La présente invention concerne le domaine des ARN thérapeutiques pour le traitement de sujets qui ont échoué, ou qui deviennent intolérants, résistants ou réfractaires à une thérapie de mort cellulaire anti-programmée 1 (PD-1) ou au ligand 1 de mort cellulaire anti-programmé 1 (PD-L1), y compris la thérapie PD-1 et/ou PD-L1 innée et acquise, ainsi que chez des sujets inopérables, présentant un stade avancé ou présentant des cancers métastatiques à tumeurs solides avec ou sans défaillance, intolérance, résistance ou réfraction à une thérapie de mort cellulaire anti-programmée 1 (PD-1) ou au ligand 1 de mort cellulaire anti-programmé 1 (PD-L1).
EP20705267.1A 2019-01-21 2020-01-17 Arn thérapeutique et anticorps anti-pd1 pour des cancers à tumeurs solides de stade avancé Pending EP3914355A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US201962794896P 2019-01-21 2019-01-21
US201962926379P 2019-10-25 2019-10-25
EP19306471 2019-11-14
PCT/US2020/014039 WO2020154189A1 (fr) 2019-01-21 2020-01-17 Arn thérapeutique et anticorps anti-pd1 pour des cancers à tumeurs solides de stade avancé

Publications (1)

Publication Number Publication Date
EP3914355A1 true EP3914355A1 (fr) 2021-12-01

Family

ID=69582159

Family Applications (1)

Application Number Title Priority Date Filing Date
EP20705267.1A Pending EP3914355A1 (fr) 2019-01-21 2020-01-17 Arn thérapeutique et anticorps anti-pd1 pour des cancers à tumeurs solides de stade avancé

Country Status (13)

Country Link
US (1) US20220288199A1 (fr)
EP (1) EP3914355A1 (fr)
JP (1) JP2022518236A (fr)
KR (1) KR20210118870A (fr)
CN (1) CN113507963A (fr)
AU (1) AU2020210614A1 (fr)
BR (1) BR112021013965A2 (fr)
CA (1) CA3126110A1 (fr)
IL (1) IL284645A (fr)
MX (1) MX2021008605A (fr)
SG (1) SG11202107396RA (fr)
TW (1) TW202043274A (fr)
WO (1) WO2020154189A1 (fr)

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BR112019017743A2 (pt) 2017-02-28 2020-04-07 Sanofi Sa rna terapêutico
TW202043256A (zh) 2019-01-10 2020-12-01 美商健生生物科技公司 前列腺新抗原及其用途
AU2020211584A1 (en) * 2019-01-21 2021-08-26 Sanofi Therapeutic RNA for advanced stage solid tumor cancers
JP2023542490A (ja) * 2020-09-03 2023-10-10 リジェネロン・ファーマシューティカルズ・インコーポレイテッド Ｐｄ－１阻害剤を投与することによりがん疼痛を処置する方法
WO2022090203A1 (fr) * 2020-10-26 2022-05-05 Cytune Pharma AGONISTE IL-2/IL-15RβҮ POUR TRAITER LE CARCINOME MALPIGHIEN
WO2022090202A1 (fr) * 2020-10-26 2022-05-05 Cytune Pharma AGONISTE D'IL-2/IL-15RBβҮ POUR LE TRAITEMENT DU CANCER DE LA PEAU NON MÉLANOME
WO2023279068A1 (fr) * 2021-07-02 2023-01-05 Gigagen, Inc. Protéines de liaison anti-ctla-4 et leurs méthodes d'utilisation
CN115260264B (zh) * 2022-02-28 2023-06-09 广州市恒诺康医药科技有限公司 用于rna加帽的化合物及其应用

Family Cites Families (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
EP2418278A3 (fr) 2005-05-09 2012-07-04 Ono Pharmaceutical Co., Ltd. Anticorps monoclonaux humains pour mort programmée 1 (PD-1) et procédés de traitement du cancer à l'aide d'anticorps anti-PD-1 seuls ou combinés à d'autres formulations immunothérapeutiques
EP3222634A1 (fr) 2007-06-18 2017-09-27 Merck Sharp & Dohme B.V. Anticorps dirigés contre le récepteur humain de mort programmée pd-1
RS54233B1 (en) 2008-08-25 2015-12-31 Amplimmune Inc. PD-1 ANTAGONIST COMPOSITIONS AND PROCEDURES FOR THEIR APPLICATION
PL2699264T3 (pl) 2011-04-20 2018-08-31 Medimmune, Llc Przeciwciała i inne cząsteczki wiążące B7-H1 i PD-1
SI2992017T1 (sl) 2013-05-02 2021-04-30 Anaptysbio, Inc. Protitelesa, usmerjena proti programirani smrti-1 (PD-1)
CN104250302B (zh) 2013-06-26 2017-11-14 上海君实生物医药科技股份有限公司 抗pd‑1抗体及其应用
CN108715615B (zh) 2013-09-13 2020-11-27 百济神州(广州)生物科技有限公司 抗pd1抗体及其作为治疗剂与诊断剂的用途
MY184154A (en) 2013-12-12 2021-03-23 Shanghai hengrui pharmaceutical co ltd Pd-1 antibody, antigen-binding fragment thereof, and medical application thereof
TWI681969B (zh) 2014-01-23 2020-01-11 美商再生元醫藥公司 針對ｐｄ-１的人類抗體
JOP20200094A1 (ar) 2014-01-24 2017-06-16 Dana Farber Cancer Inst Inc جزيئات جسم مضاد لـ pd-1 واستخداماتها
WO2016005004A1 (fr) 2014-07-11 2016-01-14 Biontech Rna Pharmaceuticals Gmbh Stabilisation de séquences d'adn codant pour une séquence poly (a)
TWI595006B (zh) 2014-12-09 2017-08-11 禮納特神經系統科學公司 抗ｐｄ－１抗體類和使用彼等之方法
JO3736B1 (ar) 2015-07-30 2021-01-31 Macrogenics Inc جزيئات رابطة لـ pd-1 وطرق استخدامها
WO2017024465A1 (fr) 2015-08-10 2017-02-16 Innovent Biologics (Suzhou) Co., Ltd. Anticorps anti-pd-1
CN107949573B (zh) 2015-09-01 2022-05-03 艾吉纳斯公司 抗-pd-1抗体及其使用方法
LT3370768T (lt) 2015-11-03 2022-05-25 Janssen Biotech, Inc. Antikūnai, specifiškai surišantys pd-1, ir jų panaudojimas
CN109715205A (zh) * 2016-08-19 2019-05-03 库瑞瓦格股份公司 用于癌症治疗的rna
BR112019017743A2 (pt) * 2017-02-28 2020-04-07 Sanofi Sa rna terapêutico

Also Published As

Publication number Publication date
SG11202107396RA (en) 2021-08-30
WO2020154189A1 (fr) 2020-07-30
TW202043274A (zh) 2020-12-01
US20220288199A1 (en) 2022-09-15
IL284645A (en) 2021-08-31
KR20210118870A (ko) 2021-10-01
CA3126110A1 (fr) 2020-07-30
BR112021013965A2 (pt) 2021-09-21
JP2022518236A (ja) 2022-03-14
CN113507963A (zh) 2021-10-15
AU2020210614A1 (en) 2021-08-26
MX2021008605A (es) 2021-10-26

Similar Documents

Publication Publication Date Title
US20220288199A1 (en) Therapeutic RNA and Anti-PD1 Antibodies for Advanced Stage Solid Tumor Cancers
JP7480248B2 (ja) 腫瘍を処置するための抗ｌａｇ－３抗体と抗ｐｄ－１抗体との組合せ
US20210040216A1 (en) Methods of treating her2-positive cancer
JP2022068355A (ja) 血液悪性腫瘍を処置するための抗ｌａｇ－３抗体
US11572405B2 (en) Combination therapy with anti-IL-8 antibodies and anti-PD-1 antibodies for treating cancer
US20230131598A1 (en) Combination treatment for cancer
WO2020212949A1 (fr) Méthodes de traitement du cancer du rein avec un anticorps anti-psma/cd3
US20220040218A1 (en) Therapeutic RNA for Advanced Stage Solid Tumor Cancers
EP3880709A1 (fr) Schéma posologique d'un anticorps anti-lag3 et polythérapie avec un anticorps anti-pd-1 pour le traitement du cancer
WO2023192478A1 (fr) Polythérapie avec des anticorps anti-il-8 et des anticorps anti-pd-1 pour le traitement du cancer
JP2024513247A (ja) 抗ｐｄ－１抗体の皮下投与によるがんの治療方法
Sledge Jr IRB-37299 BRS0070 NCT03364348
EP4136113A1 (fr) Traitement combiné pour le cancer impliquant des anticorps anti-icos et anti-pd1, impliquant éventuellement en outre des anticorps anti-tim3
WO2024050466A1 (fr) Procédés de traitement du cancer du poumon non à petites cellules à l'aide d'agents ciblés sur le facteur de transition épithélial mésenchymateux (met)
Dahlberg et al. A Phase 2 Study of Durvalumab in Combination with Tremelimumab in Malignant Pleural Mesothelioma Coordinating Center: Dana-Farber Cancer Institute
Cercek et al. Phase II Study to Evaluate the Efficacy of MEDI4736 in Imm Advanced Colorectal Cancer

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: UNKNOWN

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20210818

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

DAV Request for validation of the european patent (deleted)
DAX Request for extension of the european patent (deleted)
REG Reference to a national code

Ref country code: HK

Ref legal event code: DE

Ref document number: 40063162

Country of ref document: HK